Experimental generation of carcinoma-associated fibroblasts (CAFs) from human mammary fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes (1-3). The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of α-smooth muscle actin (α-SMA) expression(4,5). These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues (6). The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients. Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis (7-10). CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model. In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance (11). We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.     

Treatment of hypoxia‐dependent cardiovascular diseases by myo‐inositol trispyrophosphate (ITPP)‐enhancement of oxygen delivery by red blood cells

Heart failure is a consequence of progression hypoxia‐dependent tissue damages. Therapeutic approaches to restore and/or protect the healthy cardiac tissue have largely failed and remain a major challenge of regenerative medicine. The myo‐inositol trispyrophosphate (ITPP) is a modifier of haemoglobin which enters the red blood cells and modifies the haemoglobin properties, allowing for easier and better delivery of oxygen by the blood. Here, we show that this treatment approach in an in vivo model of myocardial infarction (MI) results in an efficient protection from heart failure, and we demonstrate the recovery effect on post‐MI left ventricular remodelling in the rat model. Cultured cardiomyocytes used to study the molecular mechanism of action of ITPP in vitro displayed the fast stimulation of HIF‐1 upon hypoxic conditions. HIF‐1 overexpression was prevented by ITPP when incorporated into red blood cells applied in a model of blood‐perfused cardiomyocytes coupling the dynamic shear stress effect to the enhanced O₂ supply by modification of haemoglobin ability to release O₂ in hypoxia. ITPP treatment appears a breakthrough strategy for the efficient and safe treatment of hypoxia‐ or ischaemia‐induced injury of cardiac tissue.     

Lymphotoxin Signaling Is Initiated by the Viral Polymerase in HCV-linked Tumorigenesis

Exposure to hepatitis C virus (HCV) typically results in chronic infection that leads to progressive liver disease ranging from mild inflammation to severe fibrosis and cirrhosis as well as primary liver cancer. HCV triggers innate immune signaling within the infected hepatocyte, a first step in mounting of the adaptive response against HCV infection. Persistent inflammation is strongly associated with liver tumorigenesis. The goal of our work was to investigate the initiation of the inflammatory processes triggered by HCV viral proteins in their host cell and their possible link with HCV-related liver cancer. We report a dramatic upregulation of the lymphotoxin signaling pathway and more specifically of lymphotoxin-β in tumors of the FL-N/35 HCV-transgenic mice. Lymphotoxin expression is accompanied by activation of NF-κB, neosynthesis of chemokines and intra-tumoral recruitment of mononuclear cells. Spectacularly, IKKβ inactivation in FL-N/35 mice drastically reduces tumor incidence. Activation of lymphotoxin-β pathway can be reproduced in several cellular models, including the full length replicon and HCV-infected primary human hepatocytes. We have identified NS5B, the HCV RNA dependent RNA polymerase, as the viral protein responsible for this phenotype and shown that pharmacological inhibition of its activity alleviates activation of the pro-inflammatory pathway. These results open new perspectives in understanding the inflammatory mechanisms linked to HCV infection and tumorigenesis.     

Carcinoma‐associated fibroblasts: Non‐neoplastic tumour‐promoting mesenchymal cells

Cancerous stroma coevolves alongside tumour progression, thereby promoting the malignant conversion of epithelial carcinoma cells. To date, an abundance of data have supported crucial roles of the tumour microenvironment (TME) in providing cancer cells with proliferative, migratory, survival and invasive propensities favouring the processes of tumourigenesis. The cancerous reactive stroma is frequently populated by a large number of myofibroblasts (MFs), which are activated, non‐transformed fibroblasts expressing α‐smooth muscle actin (α‐SMA). MFs together with non‐MF cells present in the tumour‐associated stroma are collectively referred to as carcinoma‐associated fibroblasts (CAFs), one of the major stromal cell types recognised in various human carcinomas. Recruitment of fibroblasts and/or their progenitors to a tumour mass and their subsequent transdifferentiation into MFs, as well as ongoing maintenance of their activated state, are believed to be essential processes facilitating tumour progression. However, the complex networks of signalling pathways mediating the phenotypic conversion into CAFs, as well as those underlying their tumour‐promoting interactions with other tumour‐constituting cells, have yet to be fully explored. Histopathological confirmation of the presence of large numbers of CAF MFs within TME and their altered gene expression profiles are known to be associated with disease progression and to serve as independent negative prognostic factors for a wide range of tumour types. In this review, we examine the current evidence shedding light on the emerging roles of tumour‐promoting CAFs, cells that are pivotal for epithelial cancer development and progression, and discuss the therapeutic potential of targeting these cells. J. Cell. Physiol. 228: 1651–1657, 2013. © 2013 Wiley Periodicals, Inc.     

The Quantum Casimir Effect May Be a Universal Force Organizing the Bilayer Structure of the Cell Membrane

A mathematic–physical model of the interaction between cell membrane bilayer leaflets is proposed based on the Casimir effect in dielectrics. This model explains why the layers of a lipid membrane gently slide one past another rather than penetrate each other. The presented model reveals the dependence of variations in the free energy of the system on the membrane thickness. This function is characterized by the two close minima corresponding to the different levels of interdigitation of the lipids from neighbor layers. The energy barrier of the compressing transition between the predicted minima is estimated to be 5.7 kT/lipid, and the return energy is estimated to be 3.1 kT/lipid. The proposed model enables estimation of the value of the membrane elastic thickness modulus of compressibility, which is 1.7 × 10⁹ N/m², and the value of the interlayer friction coefficient, which is 1.9 × 10⁸ Ns/m³.     

Dormancy removal of apple seeds by cold stratification is associated with fluctuation in H2O2, NO production and protein carbonylation level

Reactive oxygen (ROS) and nitrogen (RNS) species play a signaling role in seed dormancy alleviation and germination. Their action may be described by the oxidative/nitrosative “window/door”. ROS accumulation in embryos could lead to oxidative modification of protein through carbonylation. Mature apple (Malus domestica Borkh.) seeds are dormant and do not germinate. Their dormancy may be overcome by 70–90 days long cold stratification. The aim of this work was to analyze the relationship between germinability of embryos isolated from cold (5 °C) or warm (25 °C) stratified apple seeds and ROS or nitric oxide (NO) production and accumulation of protein carbonyl groups. A biphasic pattern of variation in H₂O₂ concentration in the embryos during cold stratification was detected. H₂O₂ content increased markedly after 7 days of seeds imbibition at 5 °C. After an additional two months of cold stratification, the H₂O₂ concentration in embryos reached the maximum. NO production by the embryos was low during entire period of stratification, but increased significantly in germination sensu stricto (i.e. phase II of the germination process). The highest content of protein carbonyl groups was detected after 6 weeks of cold stratification treatment. Fluctuation of H₂O₂ and protein carbonylation seems to play a pivotal role in seed dormancy alleviation by cold stratification, while NO appears to be necessary for seed germination.     

Quantitative Analysis of Porcine Endogenous Retroviruses in Different Organs of Transgenic Pigs Generated for Xenotransplantation

The pig appears to be the most promising animal donor of organs for use in human recipients. Among several types of pathogens found in pigs, one of the greatest problems is presented by porcine endogenous retroviruses (PERVs). Screening of the source pig herd for PERVs should include analysis of both PERV DNA and RNA. Therefore, the present study focuses on quantitative analysis of PERVs in different organs such as the skin, heart, muscle, and liver and blood of transgenic pigs generated for xenotransplantation. Transgenic pigs were developed to express the human α-galactosidase, the human α-1,2-fucosyltransferase gene, or both genetic modifications of the genome (Lipinski et al., Medycyna Wet 66:316–322, 2010; Lipinski et al., Ann Anim Sci 12:349–356, 2012; Wieczorek et al., Medycyna Wet 67:462–466, 2011). The copy numbers of PERV DNA and RNA were evaluated using real-time Q-PCR and QRT-PCR, respectively. Comparative analysis of all PERV subtypes revealed the following relationships: PERV A > PERV B > PERV C. PERV A and B were found in all samples, whereas PERV C was detected in 47 % of the tested animals. The lowest level of PERV DNA was shown in the muscles for PERV A and B and in blood samples for PERV C. The lowest level of PERV A RNA was found in the skin, whereas those of PERV B and C RNA were found in liver specimens. Quantitative analysis revealed differences in the copy number of PERV subtypes between various organs of transgenic pigs generated for xenotransplantation. Our data support the idea that careful pig selection for organ donation with low PERV copy number may limit the risk of retrovirus transmission to the human recipients.     

Growth,Metabolite Profile,Oxidative Status,and Phytohormone Levels in the Green Alga <Emphasis Type="Italic">Acutodesmus obliquus</Emphasis> Exposed to Exogenous Auxins and Cytokinins

The effects of auxins and cytokinins at the range of concentrations 0.0001–100 µM on Acutodesmus obliquus (Chlorophyceae) cultures were studied. Microalga exhibited sensitivity to cytokinins in the following order: 0.01 µM tZ?>?0.1 µM Kin?>?1 µM DPU, whereas the hierarchy of auxin activity was: 0.01 µM IAA?>?0.1 µM IBA?>?0.1 µM PAA. Cytokinins possessed higher stimulating properties on the cell number, whereas auxins increased the size of cells. Differences in the metabolite profiles of the cultures treated with phytohormones were observed. Auxins and cytokinins had a positive effect on the photosynthetic apparatus enhancing the level of chlorophylls, carotenes, and xanthophylls. In comparison with auxins, cytokinins more effectively delayed oxidative damage by increasing the level of non-enzymatic antioxidants (ascorbate, glutathione) and the activity of enzymes scavenging reactive oxidative species (catalase, glutathione reductase, ascorbate peroxidase). On the other hand, auxins stimulated superoxide dismutase activity and provoked hydrogen peroxide generation, which may be involved in cell enlargement. All phytohormones reduced the content of abscisic acid and controlled the level of endogenous auxin and cytokinins suggesting complex interactions. Different dynamics of A. obliquus responses to auxins and cytokinins clearly demonstrated their diverse roles in algal growth and metabolism.     

The combined effect of temperature and salinity changes on osmoregulation and haemocyanin concentration in Saduria entomon (Linnaeus, 1758)

Global warming is having an impact on the temperature and salinity of Baltic Sea waters. Therefore, it is important to determine the conditions in which animals can exist and how these changes may influence their functioning. Hence, the purpose of this research was to determine the broad tolerance limits of temperature and salinity of the glacial relict Saduria entomon by studying its behaviour, osmoregulatory ability and haemocyanin concentration. This effect of temperature was confirmed in the laboratory for individuals acclimated to different salinity and temperature regimes. Changes in the physiological parameters of S. entomon at various temperatures (5.5–21.5°C) and salinity levels (1–15 PSU) were recorded. There were statistically significant differences in haemolymph osmotic pressure under the influence of salinity and temperature. The mean haemolymph osmotic pressures were the lowest at 1 PSU at all the temperatures examined and the highest at 15 PSU and high temperatures 16.5 and 21.5°C. The haemocyanin concentration decreased significantly with increasing temperature at 1 PSU. There was a significant difference in haemocyanin concentration due to salinity at temperatures of 5.5 and 10.0°C (the haemocyanin concentration decreased with increasing salinity). The results showed that, although S. entomon is classified as a cold-water animal, it can survive at high temperatures above 16.5°C at least for a short time, as it is capable of osmoregulation. The tolerance to temperature changes was better than expected.