SummaryDuring 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-β-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.

The isotropic or anisotropic organisation 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 organisation. Combined experimentation and adaptation of Vicsek modelling demonstrates that the reorientation of cells relative to each other upon 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 localises to cell-cell collision zones where it locally down-regulates actomyosin activity. Without this mechanism in place cell collision guidance fails leading to isotropic matrix generation. Cross-referencing alignment and TFAP2C gene expression signatures against existing datasets enables the identification and validation of several classes of pharmacological agents that disrupt matrix anisotropy. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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.

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BackgroundGSAO (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.ResultsThe 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.ConclusionsThe 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.

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.

Traumatic brain injury is a major public health concern and is characterised by both apoptotic and necrotic cell death in the lesion. Anatomical imaging is usually used to assess traumatic brain injuries and there is a need for imaging modalities that provide complementary cellular information. We sought to non-invasively image cell death in a mouse model of traumatic brain injury using a near-infrared fluorescent conjugate of a synthetic heat shock protein-90 alkylator, 4-(N-(S-glutathionylacetyl) amino) phenylarsonous acid (GSAO). GSAO labels both apoptotic and necrotic cells coincident with loss of plasma membrane integrity. The optical GSAO specifically labelled apoptotic and necrotic cells in culture and did not accumulate in healthy organs or tissues in the living mouse body. The conjugate is a very effective imager of cell death in brain lesions. The optical GSAO was detected by fluorescence intensity and GSAO bound to dying/dead cells was detected from prolongation of the fluorescence lifetime. An optimal signal-to-background ratio was achieved as early as 3 h after injection of the probe and the signal intensity positively correlated with both lesion size and probe concentration. This optical GSAO offers a convenient and robust means to non-invasively image apoptotic and necrotic cell death in brain and other lesions.

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.