Oncogenic Ras deregulates cell-substrate interactions during mitotic rounding and respreading to alter cell division orientation

Oncogenic Ras has been shown to change the way cancer cells divide by increasing the forces generated during mitotic rounding. In this way, RasV12 enables cancer cells to divide across a wider range of mechanical environments than normal cells. Here, we identify a further role for oncogenic Ras-ERK signaling in division by showing that RasV12 expression alters the shape, division orientation, and respreading dynamics of cells as they exit mitosis. Many of these effects appear to result from the impact of RasV12 signaling on actomyosin contractility, because RasV12 induces the severing of retraction fibers that normally guide spindle positioning and provide a memory of the interphase cell shape. In support of this idea, the RasV12 phenotype is reversed by inhibition of actomyosin contractility and can be mimicked by the loss of cell-substrate adhesion during mitosis. Finally, we show that RasV12 activation also perturbs division orientation in cells cultured in 2D epithelial monolayers and 3D spheroids. Thus, the induction of oncogenic Ras-ERK signaling leads to rapid changes in division orientation that, along with the effects of RasV12 on cell growth and cell-cycle progression, are likely to disrupt epithelial tissue organization and contribute to cancer dissemination

Renal tumouroids: challenges of manufacturing 3D cultures from patient derived primary cells

Recent advancements in 3D in vitro culture have allowed for the development of cancer tissue models which accurately recapitulate the tumour microenvironment. Consequently, there has been increased innovation in therapeutic drug screening. While organoid cultures show great potential, they are limited by the time scale of their growth in vitro and the dependence upon commercial matrices, such as Matrigel, which do not allow for manipulations of their composition or mechanical properties. Here, we show a straightforward approach for the isolation and culture of primary human renal carcinoma cells and matched non-affected kidney. This approach does not require any specific selection for cancer cells, and allows for their direct culture in amenable 3D collagen-based matrices, with the preservation of cancer cells as confirmed by NGS sequencing. This method allows for culture of patient-derived cancer cells in 3D microenvironment, which can be used for downstream experimentation such as investigation of cell-matrix interaction or drug screening

Activation of synovial fibroblasts from patients at revision of their metal-on-metal total hip arthroplasty

BACKGROUND: The toxicity of released metallic particles generated in metal-on-metal (MoM) total hip arthroplasty (THA) using cobalt chromium (CoCr) has raised concerns regarding their safety amongst both surgeons and the public. Soft tissue changes such as pseudotumours and metallosis have been widely observed following the use of these implants, which release metallic by-products due to both wear and corrosion. Although activated fibroblasts, the dominant cell type in soft tissues, have been linked to many diseases, the role of synovial fibroblasts in the adverse reactions caused by CoCr implants remains unknown. To investigate the influence of implants manufactured from CoCr, the periprosthetic synovial tissues and synovial fibroblasts from patients with failed MoM THA, undergoing a revision operation, were analysed and compared with samples from patients undergoing a primary hip replacement, in order to elucidate histological and cellular changes. RESULTS: Periprosthetic tissue from patients with MoM implants was characterized by marked fibrotic changes, notably an increase in collagen content from less than 20% to 45-55%, an increase in α-smooth muscle actin positive cells from 4 to 9% as well as immune cells infiltration. Primary cell culture results demonstrated that MoM synovial fibroblasts have a decreased apoptosis rate from 14 to 6% compared to control synovial fibroblasts. In addition, synovial fibroblasts from MoM patients retained higher contractility and increased responsiveness to chemotaxis in matrix contraction. Their mechanical properties at a single cell level increased as observed by a 60% increase in contraction force and higher cell stiffness (3.3 kPa in MoM vs 2.18 kPa in control), as measured by traction force microscopy and atomic force microscopy. Further, fibroblasts from MoM patients promoted immune cell invasion by secreting monocyte chemoattractant protein 1 (MCP-1, CCL2) and induced monocyte differentiation, which could also be associated with excess accumulation of synovial macrophages. CONCLUSION: Synovial fibroblasts exposed in vivo to MoM THA implants that release CoCr wear debris displayed dramatic phenotypic alteration and functional changes. These findings unravelled an unexpected effect of the CoCr alloy and demonstrated an important role of synovial fibroblasts in the undesired tissue reactions caused by MoM THAs

Cobalt (II) ions and nanoparticles induce macrophage retention by ROS-mediated down-regulation of RhoA expression

The authors are grateful to the China Scholarship Council (CSC) for the support of Jing Xu

Mechanism of cobalt nanoparticle-induced cytotoxicity and inflammation in human macrophages.

The underlying mechanism of metal-on-metal (MoM) implants failure is still unclear. Cobalt could be the active agent due to its high toxicity and Co exposure can also occur in the environment and industry. Co(II) mimics hypoxia by stabilizing hypoxia-inducible factor (HIF)-1α, responsible for cellular and systematic responses to low oxygen levels. The hypothesis is that the HIF pathway plays an essential role in cobalt nanoparticle (Co-NP)-induced cytotoxicity and inflammation in the U937 cell line, monocyte-derived macrophages and alveolar macrophages. Ascorbic acid (AA) is essential in maintaining the activity of prolyl hydroxylases responsible for HIF-1α degradation and Co depletes intracellular AA. AA addition reduced the Co-NPs-induced HIF response and prevented Co-NPs-induced cytotoxicity. AA and glutathione (GSH) prevented reactive oxygen species (ROS) formation, but GSH had no effect on cell viability. This suggests that the AA-induced mechanism of cell protection is ROS-independent. Co-NPs, but not ions, increased release of IL-1β, which was prevented by co-exposure to AA. Caspase-1 is essential in cleaving the pro-IL-1β into its active form and AA can affect caspases activity. Preliminary results showed that AA did not affect caspase-1 suggesting a caspase-independent pathway, but further tests are required. Lathe and hip simulator CoCr-NPs stabilized HIF-1α, caused cytotoxicity and an increase in inflammatory cytokines. The lathe CoCr-NPs were more cytotoxic after 6 hours of exposure, while the hip simulator CoCr-NPs were most toxic after 24 hours. GSH was more protective than AA. MoM periprosthetic tissue showed inflammation surrounding wear debris and increased gene expression of HIF target genes, which was not observed in blood samples from patients with well-functioning MoM. This study shows for the first time the potential involvement of the HIF-1α pathway in toxicity of Co-NPs via depleting intracellular AA in a ROS- and caspase-independent manner. This new knowledge could help to explain the adverse reaction observed in patients with MoM.Open Acces

Singlet oxygen formation from photoexcited P3HT:PCBM films applied in oxidation reactions

Poly(3-hexylthiophene) thin films containing carbon-based nanostructures, i.e. fullerenes such as buckminsterfullerene (C60) or phenyl-C61-butyric acid methyl ester (PCBM), or single-walled carbon nanotubes, were investigated as heterogeneous photosensitizers producing singlet oxygen (1O2) in aerated organic solvents. Thin films were deposited on borosilicate glass using spin coating and characterized by profilometry, UV-vis, Raman and XPS. Photogeneration of 1O2 was confirmed by photooxidation of 1,3-diphenylisobenzofuran and by reaction of 1,5-dihydroxynaphthalene to juglone. The photochemical efficiency of the blends was found to depend on the carbon-based photosensitizer and can be increased by varying its concentration in the poly(3-hexylthiophene) matrix

Development of Cell-Derived Matrices for Three-DimensionalIn VitroCancer Cell Models

Most morphogenetic and pathological processes are driven by cells responding to the surrounding matrix, such as its composition, architecture, and mechanical properties. Despite increasing evidence for the role of extracellular matrix (ECM) in tissue and disease development, many in vitro substitutes still fail to effectively mimic the native microenvironment. We established a novel method to produce macroscale (>1 cm) mesenchymal cell-derived matrices (CDMs) aimed to mimic the fibrotic tumor microenvironment surrounding epithelial cancer cells. CDMs are produced by human adipose mesenchymal stem cells cultured in sacrificial 3D scaffold templates of fibronectin-coated poly-lactic acid microcarriers (MCs) in the presence of macromolecular crowders. We showed that decellularized CDMs closely mimic the fibrillar protein composition, architecture, and mechanical properties of human fibrotic ECM from cancer masses. CDMs had highly reproducible composition made of collagen types I and III and fibronectin ECM with tunable mechanical properties. Moreover, decellularized and MC-free CDMs were successfully repopulated with cancer cells throughout their 3D structure, and following chemotherapeutic treatment, cancer cells showed greater doxorubicin resistance compared to 3D culture in collagen hydrogels. Collectively, these results support the use of CDMs as a reproducible and tunable tool for developing 3D in vitro cancer models.The researchers thank MICIU (BES-2016-077182, MAT2015-68906-R, RTI2018-096320-B-C21, PGC2018-097323-A-I00, DPI2017-83721-P) and the Spanish network of cell therapy (TERCEL) for financial support. Finally, the researchers also thank Programme/Generalitat de Catalunya (2017-SGR-359) and the Severo Ochoa program of the Spanish Ministry of Science and Competitiveness (grant SEV-2014-0425, 2015-2019)

Covalent Immobilization of Organic Photosensitizers on the Glass Surface: Toward the Formation of the Light-Activated Antimicrobial Nanocoating

Two highly efficient commercial organic photosensitizers—azure A (AA) and 5-(4-aminophenyl)-10,15,20-(triphenyl)porphyrin (APTPP)—were covalently attached to the glass surface to form a photoactive monolayer. The proposed straightforward strategy consists of three steps, i.e., the initial chemical grafting of 3-aminopropyltriethoxysilane (APTES) followed by two chemical postmodification steps. The chemical structure of the resulting mixed monolayer (MIX_TC_APTES@glass) was widely characterized by X-ray photoelectron (XPS) and Raman spectroscopies, while its photoactive properties were investigated in situ by UV–Vis spectroscopy with α-terpinene as a chemical trap. It was shown that both photosensitizers retain their activity toward light-activated generation of reactive oxygen species (ROS) after immobilization on the glassy surface and that the resulting nanolayer shows high stability. Thanks to the complementarity of the spectral properties of AA and APTPP, the effectiveness of the ROS photogeneration under broadband illumination can be optimized. The reported light-activated nanocoating demonstrated promising antimicrobial activity toward Escherichia coli (E. coli), by reducing the number of adhered bacteria compared to the unmodified glass surface

Electrochemically deposited poly(selenophene)-fullerene photoactive layer: Tuning of the spectroscopic properties towards visible light-driven generation of singlet oxygen

A selenophene-containing fullerene dyad (C60Se) was electrochemically co-deposited with bis-selenophene (BisSe) to form a visible light absorbing poly(selenophene) layer with incorporated fullerene photosensitizers on platinum (Pt) or indium-tin oxide (ITO) substrates. The resulting photoactive films (P(C60Se_BisSe)) were characterized by cyclic voltammetry, UV–Vis, IR, Raman and X-ray photoelectron spectroscopies. The efficiency of P(C60Se_BisSe) towards singlet oxygen photogeneration was investigated by applying reactions with chemical traps, i.e. α-terpinene and 1,3–diphenylisobenzofuran (DPBF), monitored by UV–Vis spectroscopy. The composition of the electropolymerized layer was controlled by varying the monomers ratio in the feed solution and it had a strong influence on the spectroscopic and photosensitizing properties of the deposited film. It has been shown that the efficiency of the visible light-driven singlet oxygen generation can be increased by optimizing the ratio between C60 photosensitizers and organic units in the layer