Colon cancer cell-derived 12(S)-HETE induces the retraction of cancer-associated fibroblast via MLC2, RHO/ROCK and Ca2+ signalling

Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca2+ levels were measured and pharmacological-or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca2+, Ca(2+)calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca2+ release. Thus, Ca2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Immune Response against ALK in Children with ALK-Positive Anaplastic Large Cell Lymphoma

Patients with anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) mount a humoral and cellular immune response against ALK. More than 90% of children and adolescents with ALK-positive ALCL have detectable anti-ALK antibodies in serum or plasma, and the antibody titer inversely correlates with the risk of relapse. ALK-specific CD8 and CD4 T cell responses have been described in patients with ALK-positive ALCL. Vaccination with ALK DNA led to protection against lymphoma growth in a murine model. Collectively, these data suggest that the ALK-specific immune response is involved in the control of the disease. The characteristics of the humoral and cellular immune response against ALK as well as tumor immune escape mechanisms have been increasingly investigated. However, tumor and host factors contributing to the individual immune response against ALK are still largely unknown. Depending on the individual strength of the immune response and its determinants, individualized immunological approaches might be appropriate for the consolidation of ALCL patients. Strategies such as ALK vaccination could be effective for those with a pre-existing anti-tumor immunity, while an allogeneic blood stem cell transplantation or check-point inhibition could be effective for others

Immune Response against ALK in Children with ALK-Positive Anaplastic Large Cell Lymphoma

Patients with anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) mount a humoral and cellular immune response against ALK. More than 90% of children and adolescents with ALK-positive ALCL have detectable anti-ALK antibodies in serum or plasma, and the antibody titer inversely correlates with the risk of relapse. ALK-specific CD8 and CD4 T cell responses have been described in patients with ALK-positive ALCL. Vaccination with ALK DNA led to protection against lymphoma growth in a murine model. Collectively, these data suggest that the ALK-specific immune response is involved in the control of the disease. The characteristics of the humoral and cellular immune response against ALK as well as tumor immune escape mechanisms have been increasingly investigated. However, tumor and host factors contributing to the individual immune response against ALK are still largely unknown. Depending on the individual strength of the immune response and its determinants, individualized immunological approaches might be appropriate for the consolidation of ALCL patients. Strategies such as ALK vaccination could be effective for those with a pre-existing anti-tumor immunity, while an allogeneic blood stem cell transplantation or check-point inhibition could be effective for others

Total-Evidence Dating under the Fossilized Birth–Death Process

Bayesian total-evidence dating involves the simultaneous analysis of morphological data from the fossil record and morphological and sequence data from recent organisms, and it accommodates the uncertainty in the placement of fossils while dating the phylogenetic tree. Due to the flexibility of the Bayesian approach, total-evidence dating can also incorporate additional sources of information. Here, we take advantage of this and expand the analysis to include information about fossilization and sampling processes. Our work is based on the recently described fossilized birth–death (FBD) process, which has been used to model speciation, extinction, and fossilization rates that can vary over time in a piecewise manner. So far, sampling of extant and fossil taxa has been assumed to be either complete or uniformly at random, an assumption which is only valid for a minority of data sets. We therefore extend the FBD process to accommodate diversified sampling of extant taxa, which is standard practice in studies of higher-level taxa. We verify the implementation using simulations and apply it to the early radiation of Hymenoptera (wasps, ants, and bees). Previous total-evidence dating analyses of this data set were based on a simple uniform tree prior and dated the initial radiation of extant Hymenoptera to the late Carboniferous (309 Ma). The analyses using the FBD prior under diversified sampling, however, date the radiation to the Triassic and Permian (252 Ma), slightly older than the age of the oldest hymenopteran fossils. By exploring a variety of FBD model assumptions, we show that it is mainly the accommodation of diversified sampling that causes the push toward more recent divergence times. Accounting for diversified sampling thus has the potential to close the long-discussed gap between rocks and clocks. We conclude that the explicit modeling of fossilization and sampling processes can improve divergence time estimates, but only if all important model aspects, including sampling biases, are adequately addressed.This article is from Syst Biol (2016) 65 (2):228-249. doi:10.1093/sysbio/syv080. Posted with permission.</p

Total-evidence dating under the fossilized birth-death process

Bayesian total-evidence dating involves the simultaneous analysis of morphological data from the fossil record and morphological and sequence data from recent organisms, and it accommodates the uncertainty in the placement of fossils while dating the phylogenetic tree. Due to the flexibility of the Bayesian approach, total-evidence dating can also incorporate additional sources of information. Here, we take advantage of this and expand the analysis to include information about fossilization and sampling processes. Our work is based on the recently described fossilized birth–death (FBD) process, which has been used to model speciation, extinction, and fossilization rates that can vary over time in a piecewise manner. So far, sampling of extant and fossil taxa has been assumed to be either complete or uniformly at random, an assumption which is only valid for a minority of data sets. We therefore extend the FBD process to accommodate diversified sampling of extant taxa, which is standard practice in studies of higher-level taxa. We verify the implementation using simulations and apply it to the early radiation of Hymenoptera (wasps, ants, and bees). Previous total-evidence dating analyses of this data set were based on a simple uniform tree prior and dated the initial radiation of extant Hymenoptera to the late Carboniferous (309 Ma). The analyses using the FBD prior under diversified sampling, however, date the radiation to the Triassic and Permian (252 Ma), slightly older than the age of the oldest hymenopteran fossils. By exploring a variety of FBD model assumptions, we show that it is mainly the accommodation of diversified sampling that causes the push toward more recent divergence times. Accounting for diversified sampling thus has the potential to close the long-discussed gap between rocks and clocks. We conclude that the explicit modeling of fossilization and sampling processes can improve divergence time estimates, but only if all important model aspects, including sampling biases, are adequately addressed

Intravasation of SW620 colon cancer cell spheroids through the blood endothelial barrier is inhibited by clinical drugs and flavonoids in vitro

Mechanisms how colorectal cancer (CRC) cells penetrate blood micro-vessel endothelia and metastasise is poorly understood. To study blood endothelial cell (BEC) barrier breaching by CRC emboli, an in vitro assay measuring BEC-free areas underneath SW620 cell spheroids, so called "circular chemorepellent induced defects" (CCIDs, appearing in consequence of endothelial retraction), was adapted and supported by Western blotting, EIA-, EROD- and luciferase reporter assays. Inhibition of ALOX12 or NF-kappa B in SW620 cells or BECs, respectively, caused attenuation of CCIDs. The FDA approved drugs vinpocetine [inhibiting ALOX12-dependent 12(S)-HETE synthesis], ketotifen [inhibiting NF-kappa B], carbamazepine and fenofibrate [inhibiting 12(S)-HETE and NF-kappa B] significantly attenuated CCID formation at low mu M concentrations. In the 5-FU-resistant SW620-R/BEC model guanfacine, nifedipine and proadifen inhibited CCIDs stronger than in the naive SW620/BEC model. This indicated that in SW620-R cells formerly silent (yet unidentified). genes became expressed and targetable by these drugs in course of resistance acquisition. Fenofibrate, and the flavonoids hispidulin and apigenin, which are present in medicinal plants, spices, herbs and fruits, attenuated CCID formation in both, naive- and resistant models. As FDA-approved drugs and food-flavonoids inhibited established and acquired intravasative pathways and attenuated BEC barrier-breaching in vitro, this warrants testing of these compounds in CRC models in vivo