Tumor-induced remote ECM network orientation steers angiogenesis

Tumor angiogenesis promotes tumor growth and metastasis. Here, we use automated sequential microprinting of tumor and endothelial cells in extracellular matrix (ECM) scaffolds to study its mechanical aspects. Quantitative reflection microscopy shows that tumor spheroids induce radial orientation of the surrounding collagen fiber network up to a distance of five times their radius. Across a panel of ~20 different human tumor cell lines, remote collagen orientation is correlated with local tumor cell migration behavior. Tumor induced collagen orientation requires contractility but is remarkably resistant to depletion of collagen-binding integrins. Microvascular endothelial cells undergo directional migration towards tumor spheroids once they are within the tumor-oriented collagen fiber network. Laser ablation experiments indicate that an intact physical connection of the oriented network with the tumor spheroid is required for mechanical sensing by the endothelial cells. Together our findings indicate that, in conjunction with described activities of soluble angiogenic factors, remote physical manipulation of the ECM network by the tumor can help to steer angiogenesis

Cellulaire 'systeempsychologie' en geneesmiddeltoxiciteit

Populariserende publicati

Mechanistic insights in TNF signaling and drug-induced liver injury: towards a predictive preclinical toolbox

There is an increasing body of evidence that various drugs can sensitize hepatocytes to TNF-mediated killing. This interaction is likely due to perturbations of the normal hepatocyte physiology that triggers a pro-apoptotic TNFR pathway when TNF is present in the system. We have focussed on the effects of DILI compounds on endoplasmic reticulum (ER) stress as well as the Nrf2-mediated oxidative stress adaptive toxicity pathways and how these two pathways converge with adverse TNFR signaling. Moreover, we have deepened our understanding on effects of DILI compounds on the TNFR-mediated NF-kappa B regulation. We have integrated primary human hepatocyte transcriptomics data with live cell imaging adaptive stress response GFP-reporter data to unravel in detail the interaction between different adaptive stress pathways and TNF/drug cytotoxic synergy. Moreover we have applied RNA interference screening to identify key modulators of these signaling pathways that define this synergistic toxic interaction. Our data provide suggestions on the toolbox components that can be used in a preclinical drug safety testing phase to assess safety liabilities for a drug/cytokine interplay in the development of DILI. This work is part of the MIP-DILI project supported by the Innovative Medicines Initiative (grant agreement n° 115336), and the FP7 SEURAT-1 DETECTIVE project (grant agreement 266838).Toxicolog

A high throughput microscopy toxicity pathway reporter platform for chemical safety assessment

Toxicolog

The B∗BπB^*B\pi coupling with relativistic heavy quarks

We report on a calculation of the $B^*B\pi$ coupling in lattice QCD. The strong matrix element $\langle B \pi | B^*\rangle$ is directly related to the leading order low-energy constant in heavy meson chiral perturbation theory (HM$\chi$PT) for $B$-mesons. We carry out our calculation directly at the $b$-quark mass using a non-perturbatively tuned clover action that controls discretisation effects of order $|\vec{p}a|$ and $(ma)^n$ for all $n$. Our analysis is performed on RBC/UKQCD gauge configurations using domain wall fermions and the Iwasaki gauge action at two lattice spacings of $a^{-1}=1.73(3)$ GeV, $a^{-1}=2.28(3)$ GeV, and unitary pion masses down to 290 MeV. We achieve good statistical precision and control all systematic uncertainties, giving a final result for the HM$\chi$PT coupling $g_b = 0.569(48)_{stat}(59)_{sys}$ in the continuum and at the physical light-quark masses. This is the first calculation performed directly at the physical $b$-quark mass and lies in the region one would expect from carrying out an interpolation between previous results at the charm mass and at the static point.Comment: 7 pages, 2 figures, presented at the 31st International Symposium on Lattice Field Theory (Lattice 2013), 29 July - 3 August 2013, Mainz, German

Annexin A2 phosphorylation mediates cell scattering and branching morphogenesis via cofilin Activation

Dynamic remodeling of the actin cytoskeleton is required for cell spreading, motility, and migration and can be regulated by tyrosine kinase activity. Phosphotyrosine proteomic screening revealed phosphorylation of the lipid-, calcium-, and actin-binding protein annexin A2 (AnxA2) at Tyr23 as a major event preceding ts-v-Src kinase-induced cell scattering. Expression of the phospho-mimicking mutant Y23E-AnxA2 itself was sufficient to induce actin reorganization and cell scattering in MDCK cells. While Y23E-AnxA2, but not Y23A-AnxA2, enhanced Src- or hepatocyte growth factor (HGF)-induced cell scattering, short hairpin RNA-mediated knockdown of AnxA2 inhibited both v-Src- and HGF-induced cell scattering. Three-dimensional branching morphogenesis was induced in wild-type-AnxA2-expressing cells only in the presence of HGF, while Y23E-AnxA2 induced HGF-independent branching morphogenesis. Knockdown of AnxA2 prevented lumen formation during cystogenesis. The Y23E-AnxA2-induced scattering was associated with dephosphorylation/activation of the actin-severing protein cofilin. Likewise, inactive S3E-cofilin and constitutively active LIM kinase, a direct upstream kinase of cofilin, inhibited Y23E-AnxA2-induced scattering. Together, our studies indicate an essential role for AnxA2 phosphorylation in regulating cofilin-dependent actin cytoskeletal dynamics in the context of cell scattering and branching morphogenesis

Heterogeneity of autoreactive T cell clones specific for the E2 component of the pyruvate dehydrogenase complex in primary biliary cirrhosis.

The extraordinary specificity of bile duct destruction in primary biliary cirrhosis (PBC) and the presence of T cell infiltrates in the portal tracts have suggested that biliary epithelial cells are the targets of an autoimmune response. The immunodominant antimitochondrial response in patients with PBC is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). Hitherto, there have only been limited reports on the characterization and V beta usage of PDC-E2-specific cloned T cell lines. In this study, we examined peripheral blood mononuclear cells (PBMC) for their reactivity to the entire PDC complex as well as to the E1- and E2-specific components. We also examined the phenotype, lymphokine profile, and V beta usage of PDC-specific T cell clones isolated from cellular infiltrates from the livers of PBC patients. We report that PBMC from 16/19 patients with PBC, but not 12 control patients, respond to the PDC-E2 subunit. Interestingly, this response was directed to the inner and/or the outer lipoyl domains, despite the serologic observation that the autoantibody response is directed predominantly to the inner lipoyl domain. Additionally, lymphokine analysis of interleukin (IL) 2/IL-4/interferon gamma production from individual liver-derived autoantigen-specific T cell clones suggests that both T helper cell Th1- and Th2-like clones are present in the liver. Moreover, there was considerable heterogeneity in the T cell receptor for antigen (TCR) V beta usage of these antigen-specific autoreactive T cell clones. This is in contrast to murine studies in which animals are induced to develop autoimmunity by specific immunization and have an extremely limited T cell V beta repertoire. Thus, our data suggest that in human organ-specific autoimmune diseases, such as PBC, the TCR V beta repertoire is heterogenous

Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells

The relationship between focal adhesion protein (FAK) activity and loss of cell-matrix contact during apoptosis is not entirely clear nor has the role of FAK in chemically induced apoptosis been studied. We investigated the status of FAK phosphorylation and cleavage in renal epithelial cells during apoptosis caused by the nephrotoxicant dichlorovinylcysteine (DCVC). DCVC treatment caused a loss of cell-matrix contact which was preceded by a dissociation of FAK from the focal adhesions and tyrosine dephosphorylation of FAK. Paxillin was also dephosphorylated at tyrosine. DCVC treatment activated caspase-3 which was associated with cleavage of FAK. However, FAK cleavage occurred after cells had already lost focal adhesions indicating that cleavage of FAK by caspases is not responsible for loss of FAK from focal adhesions. Accordingly, although inhibition of caspase activity with zVAD-fmk blocked activation of caspase-3, FAK cleavage, and apoptosis, it neither affected dephosphorylation nor translocation of FAK or paxillin. However, zVAD-fmk completely blocked the cell detachment caused by DCVC treatment. Orthovanadate prevented DCVC-induced tyrosine dephosphorylation of both FAK and paxillin; however, it did not inhibit DCVC-induced apoptosis and actually potentiated focal adhesion disorganization and cell detachment. Thus, FAK dephosphorylation and loss of focal adhesions are not due to caspase activation; however, caspases are required for FAK proteolysis and cell detachment.Toxicolog

High-content imaging-based BAC-GFP toxicity pathway reporters to assess chemical adversity liabilities.

Adaptive cellular stress responses are paramount in the healthy control of cell and tissue homeostasis and generally activated during toxicity in a chemical-specific manner. Here, we established a platform containing a panel of distinct adaptive stress response reporter cell lines based on BAC-transgenomics GFP tagging in HepG2 cells. Our current panel of eleven BAC-GFP HepG2 reporters together contains (1) upstream sensors, (2) downstream transcription factors and (3) their respective target genes, representing the oxidative stress response pathway (Keap1/Nrf2/Srxn1), the unfolded protein response in the endoplasmic reticulum (Xbp1/Atf4/BiP/Chop) and the DNA damage response (53bp1/p53/p21). Using automated confocal imaging and quantitative single-cell image analysis, we established that all reporters allowed the time-resolved, sensitive and mode-of-action-specific activation of the individual BAC-GFP reporter cell lines as defined by a panel of pathway-specific training compounds. Implementing the temporal pathway activity information increased the discrimination of training compounds. For a set of >30 hepatotoxicants, the induction of Srxn1, BiP, Chop and p21 BAC-GFP reporters correlated strongly with the transcriptional responses observed in cryopreserved primary human hepatocytes. Together, our data indicate that a phenotypic adaptive stress response profiling platform will allow a high throughput and time-resolved classification of chemical-induced stress responses, thus assisting in the future mechanism-based safety assessment of chemicals.Toxicolog