SnapShot: Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) are an integral component of the tumor microenvironment and have both tumor-promoting and tumor-suppressive functions. This SnapShot summarizes the origins of CAFs, their diverse functions, and how this relates to heterogeneity within the population. The suitability of targeting CAFs therapeutically is also discussed.

Extracellular matrix anisotropy is determined by TFAP2C-dependent regulation of cell collisions.

The isotropic or anisotropic organization of biological extracellular matrices has important consequences for tissue function. We study emergent anisotropy using fibroblasts that generate varying degrees of matrix alignment from uniform starting conditions. This reveals that the early migratory paths of fibroblasts are correlated with subsequent matrix organization. Combined experimentation and adaptation of Vicsek modelling demonstrates that the reorientation of cells relative to each other following collision plays a role in generating matrix anisotropy. We term this behaviour 'cell collision guidance'. The transcription factor TFAP2C regulates cell collision guidance in part by controlling the expression of RND3. RND3 localizes to cell-cell collision zones where it downregulates actomyosin activity. Cell collision guidance fails without this mechanism in place, leading to isotropic matrix generation. The cross-referencing of alignment and TFAP2C gene expression signatures against existing datasets enables the identification and validation of several classes of pharmacological agents that disrupt matrix anisotropy.

Matrix feedback enables diverse higher-order patterning of the extracellular matrix.

The higher-order patterning of extra-cellular matrix in normal and pathological tissues has profound consequences on tissue function. Whilst studies have documented both how fibroblasts create and maintain individual matrix fibers and how cell migration is altered by the fibers they interact with, a model unifying these two aspects of tissue organization is lacking. Here we use computational modelling to understand the effect of this interconnectivity between fibroblasts and matrix at the mesoscale level. We created a unique adaptation to the Vicsek flocking model to include feedback from a second layer representing the matrix, and use experimentation to parameterize our model and validate model-driven hypotheses. Our two-layer model demonstrates that feedback between fibroblasts and matrix increases matrix diversity creating higher-order patterns. The model can quantitatively recapitulate matrix patterns of tissues in vivo. Cells follow matrix fibers irrespective of when the matrix fibers were deposited, resulting in feedback with the matrix acting as temporal 'memory' to collective behaviour, which creates diversity in topology. We also establish conditions under which matrix can be remodelled from one pattern to another. Our model elucidates how simple rules defining fibroblast-matrix interactions are sufficient to generate complex tissue patterns.

Optical imaging of treatment-related tumor cell death using a heat shock protein-90 alkylator.

The ability to assess in near-real time the tumor cell killing efficacy of chemotherapy regimens would improve patient treatment and survival. An ineffective regimen could be abandoned early in favor of a more effective treatment. We sought to noninvasively image treatment-related tumor cell death in mice using an optically labeled synthetic heat shock protein-90 (Hsp90) alkylator, 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid (GSAO). The Hsp90 chaperone is an important element in oncogene addiction and tumor cell survival, and its expression is enhanced by chemotherapy. These factors were predicted to favor the detection of tumor cell death using GSAO. GSAO specifically labeled apoptotic and necrotic tumor cells in culture and cells of comparable morphology in subcutaneous human pancreatic carcinoma tumors in mice. A near-infrared fluorescent conjugate of GSAO was used to noninvasively image cyclophosphamide-induced tumor cell death in murine orthotopic human mammary tumors. The GSAO conjugate did not accumulate in healthy organs or tissues in the mouse, and unbound compound was excreted rapidly via the kidneys. There was a significant increase in the GSAO fluorescence signal in the treated tumors measured either in vivo or ex vivo, and the fluorescence signal colocalized with apoptotic cells in sectioned tumors. The favorable biodistribution of optically labeled GSAO, the nature of its tumor cell target, and its capacity to noninvasively detect tumor cell death should facilitate the application of this compound in studies of the efficacy of existing and new chemotherapeutics.

The tumour metabolism inhibitors GSAO and PENAO react with cysteines 57 and 257 of mitochondrial adenine nucleotide translocase.

BACKGROUND: GSAO (4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid) and PENAO (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid) are tumour metabolism inhibitors that target adenine nucleotide translocase (ANT) of the inner-mitochondrial membrane. Both compounds are currently being trialled in patients with solid tumours. The trivalent arsenical moiety of GSAO and PENAO reacts with two matrix facing cysteine residues of ANT, inactivating the transporter. This leads to proliferation arrest and death of tumour and tumour-supporting cells. RESULTS: The two reactive ANT cysteine residues have been identified in this study by expressing cysteine mutants of human ANT1 in Saccharomyces cerevisiae and measuring interaction with the arsenical moiety of GSAO and PENAO. The arsenic atom of both compounds cross-links cysteine residues 57 and 257 of human ANT1. CONCLUSIONS: The sulphur atoms of these two cysteines are 20 Å apart in the crystal structures of ANT and the optimal spacing of cysteine thiolates for reaction with As (III) is 3-4 Å. This implies that a significant conformational change in ANT is required for the organoarsenicals to react with cysteines 57 and 257. This conformational change may relate to the selectivity of the compounds for proliferating cells.

Noninvasive imaging of cell death using an Hsp90 ligand.

Cell death plays a central role in normal physiology and in disease. Common to apoptotic and necrotic cell death is the eventual loss of plasma membrane integrity. We have produced a small organoarsenical compound, 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid, that rapidly accumulates in the cytosol of dying cells coincident with loss of plasma membrane integrity. The compound is retained in the cytosol predominantly by covalent reaction with the 90 kDa heat shock protein (Hsp90), the most abundant molecular chaperone of the eukaryotic cytoplasm. The organoarsenical was tagged with either optical or radioisotope reporting groups to image cell death in cultured cells and in murine tumors ex vivo and in situ. Tumor cell death in mice was noninvasively imaged by SPECT/CT using an (111)In-tagged compound. This versatile compound should enable the imaging of cell death in most experimental settings.

CAFs and Cancer Cells Co-Migration in 3D Spheroid Invasion Assay.

In many solid tumors, collective cell invasion prevails over single-cell dissemination strategies. Collective modes of invasion often display specific front/rear cellular organization, where invasive leader cells arise from cancer cell populations or the tumor stroma. Collective invasion involves coordinated cellular movements which require tight mechanical crosstalk through specific combinations of cell-cell interactions and cell-matrix adhesions. Cancer Associated Fibroblasts (CAFs) have been recently reported to drive the dissemination of epithelial cancer cells through ECM remodeling and direct intercellular contact. However, the cooperation between tumor and stromal cells remains poorly understood. Here we present a simple spheroid invasion assay to assess the role of CAFs in the collective migration of epithelial tumor cells. This method enables the characterization of 3D spheroid invasion patterns through live cell fluorescent labeling combined with spinning disc microscopy. When embedded in extracellular matrix, the invasive strands of spheroids can be tracked and leader/follower organization of CAFs and cancer cells can be quantified.

A FIJI macro for quantifying pattern in extracellular matrix.

Diverse extracellular matrix patterns are observed in both normal and pathological tissue. However, most current tools for quantitative analysis focus on a single aspect of matrix patterning. Thus, an automated pipeline that simultaneously quantifies a broad range of metrics and enables a comprehensive description of varied matrix patterns is needed. To this end, we have developed an ImageJ plugin called TWOMBLI, which stands for The Workflow Of Matrix BioLogy Informatics. This pipeline includes metrics of matrix alignment, length, branching, end points, gaps, fractal dimension, curvature, and the distribution of fibre thickness. TWOMBLI is designed to be quick, versatile and easy-to-use particularly for non-computational scientists. TWOMBLI can be downloaded from https://github.com/wershofe/TWOMBLI together with detailed documentation and tutorial video. Although developed with the extracellular matrix in mind, TWOMBLI is versatile and can be applied to vascular and cytoskeletal networks. Here we present an overview of the pipeline together with examples from a wide range of contexts where matrix patterns are generated.

Neuron-Specific IMP2 Overexpression by Synapsin Promoter-Driven AAV9: A Tool to Study Its Role in Axon Regeneration.

Insulin-like growth factor II mRNA-binding protein (IMP) 2 is one of the three homologues (IMP1-3) that belong to a conserved family of mRNA-binding proteins. Its alternative splice product is aberrantly expressed in human hepatocellular carcinoma, and it is therefore identified as HCC. Previous works have indicated that IMP1/ZBP1 (zipcode binding protein) is critical in axon guidance and regeneration by regulating localization and translation of specific mRNAs. However, the role of IMP2 in the nervous system is largely unknown. We used the synapsin promoter-driven adeno-associated viral (AAV) 9 constructs for transgene expression both in vitro and in vivo. These viral vectors have proven to be effective to transduce the neuron-specific overexpression of IMP2 and HCC. Applying this viral vector in the injury-conditioned dorsal root ganglion (DRG) culture demonstrates that overexpression of IMP2 significantly inhibits axons regenerating from the neurons, whereas overexpression of HCC barely interrupts the process. Quantitative analysis of binding affinities of IMPs to ß-actin mRNA reveals that it is closely associated with their roles in axon regeneration. Although IMPs share significant structural homology, the distinctive functions imply their different ability to localize specific mRNAs and to regulate the axonal translation.

Immunotherapy: breaching the barriers for cancer treatment.

The great ambition to treat cancer through harnessing a patient's own immune responses has started to become reality. Clinical trials have shown impressive results and some patients reaching the end of existing treatment options have achieved full remission. Yet the response rate even within the most promising trials remain at just 30-40% of patients. To date, the focus of immunotherapy research has been to identify tumour antigens, and to enhance activation of effector lymphocytes. Yet this is only the first step to effective immunotherapy for a broader range of patients. Activated cytotoxic T cells can only act on their tumour cell targets if they have free and easy access to all tumour regions. Solid tumours are complex, heterogeneous environments which vary greatly in their physical properties. We must now focus our efforts on understanding how factors such as the composition, density and geometry of tumour extracellular matrix acts to impede or promote immune cell infiltration and activation, and work to design novel pharmacological interventions which restore and enhance leucocyte trafficking within solid tumours. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

Corrigendum: p120-catenin prevents multinucleation through control of MKLP1-dependent RhoA activity during cytokinesis.

[This corrects the article DOI: 10.1038/ncomms13874.].

p120-catenin prevents multinucleation through control of MKLP1-dependent RhoA activity during cytokinesis.

Spatiotemporal activation of RhoA and actomyosin contraction underpins cellular adhesion and division. Loss of cell-cell adhesion and chromosomal instability are cardinal events that drive tumour progression. Here, we show that p120-catenin (p120) not only controls cell-cell adhesion, but also acts as a critical regulator of cytokinesis. We find that p120 regulates actomyosin contractility through concomitant binding to RhoA and the centralspindlin component MKLP1, independent of cadherin association. In anaphase, p120 is enriched at the cleavage furrow where it binds MKLP1 to spatially control RhoA GTPase cycling. Binding of p120 to MKLP1 during cytokinesis depends on the N-terminal coiled-coil domain of p120 isoform 1A. Importantly, clinical data show that loss of p120 expression is a common event in breast cancer that strongly correlates with multinucleation and adverse patient survival. In summary, our study identifies p120 loss as a driver event of chromosomal instability in cancer.

Mesenchymal Cancer Cell-Stroma Crosstalk Promotes Niche Activation, Epithelial Reversion, and Metastatic Colonization.

During metastatic colonization, tumor cells must establish a favorable microenvironment or niche that will sustain their growth. However, both the temporal and molecular details of this process remain poorly understood. Here, we found that metastatic initiating cells (MICs) exhibit a high capacity for lung fibroblast activation as a result of Thrombospondin 2 (THBS2) expression. Importantly, inhibiting the mesenchymal phenotype of MICs by blocking the epithelial-to-mesenchymal transition (EMT)-associated kinase AXL reduces THBS2 secretion, niche-activating ability, and, consequently, metastatic competence. Subsequently, disseminated metastatic cells revert to an AXL-negative, more epithelial phenotype to proliferate and decrease the phosphorylation levels of TGF-ß-dependent SMAD2-3 in favor of BMP/SMAD1-5 signaling. Remarkably, newly activated fibroblasts promote this transition. In summary, our data reveal a crosstalk between cancer cells and their microenvironment whereby the EMT status initially triggers and then is regulated by niche activation during metastatic colonization.

A group approach to the depressed adolescent girl in foster care.

Depression reactive to severe early loss and current environmental pressure is a salient common characteristic of many adolescent girls in foster care. This paper describes work with developmentally-oriented mutual aid groups focused on depression and coping, which have been successful in promoting ego strengths and growth in this population.

Retrograde flow of cadherins in collective cell migration.

Collective cell migration is characterized by the maintenance of intercellular contacts during cell movement. The maintenance of N-cadherin-based junctions during collective migration is now shown to be facilitated by their treadmilling from the cell front to the rear, followed by N-cadherin endocytosis and recycling to the leading edge.

Influence of treatment with intranasal corticosteroids on the nasal mucosa, weight, and corticosteroid concentration in rats.

BACKGROUND: The effect of intranasal corticosteroids on the nasal epithelium mucosa is an important parameter of treatment safety. This study was designed to examine whether treatment with topical corticosteroids in patients with allergic rhinitis causes atrophic nasal mucosal changes, when compared with systemic corticosteroids, in rats. METHODS: Male Wistar rats were treated daily during 7 weeks with topical administration with 10 microliters of normal saline (control group), 10 microliters of mometasone furoate group, 10 microliters of triamcinolone acetonide (T group), and 8 mg/kg of daily subcutaneous injections of methylprednisolone sodium succinate (MP group). Body weight was evaluated weekly. At the end of the treatment, rats were killed by decapitation to collect blood for determination of corticosterone levels and nasal cavities were prepared for histological descriptive analyses. RESULTS: Treatment with T and MP decreased body weight. Plasma corticosterone concentration was significantly reduced by MP treatment and presented a clear tendency to decrease after T treatment. Histological changes observed in group T included ripples, cell vacuolization, increase in the number of nuclei, and decrease in the number of cilia in the epithelial cells. CONCLUSION: Growth and corticosterone concentration were impaired by T and MP at the same proportion, suggesting a role of this hormone in body gain. With the exception of T, intranasal or systemic treatment with the corticosteroids evaluated in this study did not affect nasal mucosa.

Mitochondria as targets in angiogenesis inhibition.

Angiogenesis is integral to the growth and metastatic spread of tumours, and its targeting is an effective anti-tumour strategy. Currently hundreds of anti-angiogenic therapeutics exist in varying stages of development, a number of which have recently gained US Food and Drug Administration (FDA) approval for the treatment of various human cancers. One class of anti-angiogenic agents directly inhibit endothelial cell function and induce endothelial cell death so as to prevent their integration into new blood vessels. The mitochondria are the focal point for a variety of pro-apoptotic signals, and this review highlights those anti-angiogenic agents that involve the mitochondria in the execution of endothelial cell death. A brief overview of angiogenesis and the mitochondrial apoptotic pathway is also given.

Análise da qualidade de adesão de diferentes bases de braquetes metálicos / Analysis of the adhesion quality of different metallic bracket bases

Diante das diferenças existentes nas características das bases dos braquetes usados atualmente, objetivou-se neste trabalho comparar entre si três tipos de bases de braquetes metálicos (Monobloc, Equilibrium e Dynalock). Foram utilizados 36 pré-molares humanos, divididos em 3 grupos de 12 dentes. Os dentes foram incluídos em troquéis com gesso pedra tipo IV e posicionados com suas faces vestibulares perpendiculares à base do troquel. Todos os braquetes foram colados com o compósito Concise Ortodôntico e submetidos ao ensaio de cisalhamento em uma Máquina Universal com uma velocidade de 0,5 mm por minuto. O braquete Monobloc obteve o maior valor médio de resistência adesiva (x = 28,19 Kgf/cm²), sendo superior estatisticamente aos braquetes Equilibrium (x = 18,07 Kgf/cm²) e Dynalock (x = 18,24 Kgf/cm²). Em relação ao ARI (Índice de Remanescente Resinoso), não foi encontrada diferença estatística entre os braquetes testados

Análise da resistência ao cisalhamento de cimentos de ionômero de vidro modificados por resina / Analisys of shear bond strength of resin-midified glass ionomer cements

O objetivo deste estudo foi comparar a resistência ao cisalhamento de bráquetes metálicos colados com os cimentos de ionômero de vidro modificados por resina Fuji Ortho LC e Ortho Glass P. Foram utilizados quarenta incisivos inferiores permanentes bovinos divididos em quatro grupos (n=10). No primeiro e segundo grupos, os bráquetes foram colados com Fuji Ortho LC sobre umidade em superfícies condicionadas e não condicionadas, respectivamente. No terceiro e quarto grupos, a colagem foi realizada com Ortho Glass P, nas mesmas condições dos grupos anteriores. Após a colagem, realizou-se o ensaio de cisalhamento de toda amostra à velocidade de 0,5 mm por minuto. Os resultados (MPa) mostraram superioridade estatística do Fuji Ortho LC em esmalte condicionado (16,514) e sem condicionamento (9,332) em relação aos grupos colados com Ortho Glass P (2,559 com condicionamento e 2,008 sem condicionamento). Entre os dois grupos colados com Ortho Glass P não houve diferença estatística significante.