Extracellular proteolysis mediates tissue homeostasis. In cancer, altered proteolysis leads to unregulated tumor growth, tissue remodeling, inflammation, tissue invasion, and metastasis. The matrix metalloproteinases (MMPs) represent the most prominent family of proteinases associated with tumorigenesis. Recent technological developments have markedly advanced our understanding of MMPs as modulators of the tumor microenvironment. In addition to their role in extracellular matrix turnover and cancer cell migration, MMPs regulate signaling pathways that control cell growth, inflammation, or angiogenesis and may even work in a nonproteolytic manner. These aspects of MMP function are reorienting our approaches to cancer therapy.

Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.Reliable visualization of the actin cytoskeleton is essential for various fields of biomedical research. Imaging of actin dynamics has been mostly achieved by injection of fluorescently labeled actin (technically demanding) or small amounts of fluorescently labeled phalloidin, an F-actin-binding and stabilizing compound 1,2 . A widely used alternative is the expression of actin-GFP fusion proteins. However, all described actin fusions are functionally impaired and rely on nontagged actin 3 to buffer the defects. Recently, fusions of GFP to actin-binding domains have been used, notably from moesin in Drosophila melanogaster 4 , LimE in Dictyostelium discoideum 5 , ABP120 in D. discoideum and mammalian cells 6,7 , and utrophin in Xenopus laevis 8 . These probes consist of large domains, compete with their endogenous counterparts and are restricted to cells that can be transfected.Abp140-GFP is the only probe that has been successfully used to label actin cables, in budding yeast 9,10 . Using total internal reflection (TIRF) microscopy to monitor localization of Abp140 domains fused to GFP, we found that the first 17 aa of Abp140 were sufficient to mediate actin Correspondence should be addressed to M.S.

Small-vessel vasculitis (SVV) is a chronic autoinflammatory condition linked to antineutrophil cytoplasm autoantibodies (ANCAs). Here we show that chromatin fibers, so-called neutrophil extracellular traps (NETs), are released by ANCA-stimulated neutrophils and contain the targeted autoantigens proteinase-3 (PR3) and myeloperoxidase (MPO). Deposition of NETs in inflamed kidneys and circulating MPO-DNA complexes suggest that NET formation triggers vasculitis and promotes the autoimmune response against neutrophil components in individuals with SVV.SVV is a relapsing-remitting autoinflammatory disorder leading to necrotic inflammation of small-sized blood vessels and capillaries 1 . ANCAs directed against granule proteins of neutrophils, namely against PR3 in Wegener's granulomatosis and MPO in microscopic polyangiitis, are implicated in the pathogenesis of SVV 2 . In vitro studies have demonstrated an activating effect of ANCAs on cytokine-primed neutrophils 3 , which was further corroborated by animal models of these diseases 4,5 . However, the basic mechanism that induces the life-threatening exacerbations of vasculitis and the sustained autoimmune response against neutrophil components remains elusive.A unique type of cell death of neutrophil granulocytes has recently been discovered that is characterized by the active release of chromatin fibers, so-called NETs, that trap and kill invading microbes extracellularly 6 . However, this glutinous DNA web can also stick to the endothelium and cause tissue damage during sepsis 7 , similar to neutrophil-induced As ANCA can activate the respiratory burst by binding to PR3 or MPO on the neutrophil surface 3 , we examined whether ANCA-mediated activation of neutrophils induces NET formation. We primed isolated neutrophils with tumor necrosis factor-α and incubated them with purified IgG from individuals with SVV or healthy donors as performed previously 3 . We observed robust NET formation (as determined by immunofluorescence 6,8; Supplementary Methods online) in neutrophils incubated with ANCA-IgG (Fig. 1a and Supplementary Table 1 online) but not in those incubated with control IgG, in which most nuclei retained the typical lobulated structure (Fig. 1b). After 180 min, we found that 23% of neutrophils incubated with ANCA-IgG produced NETs, compared to 11% of control IgG-treated neutrophils (Fig. 1c). Incubation with phorbol 12-myristate 13-acetate (PMA), known as a strong inducer of NETs, triggered NET production in 38% of all neutrophils (Fig. 1c). We also induced NETs with a PR3-specific mouse monoclonal antibody ( Supplementary Fig. 1 online), supporting the hypothesis that PR3-specific autoantibodies within the ANCA-IgG fraction trigger NET formation. ANCA-induced cell death of neutrophils was previously regarded as a dysregulated form of apoptosis 9 , but the link to NETs had not been noticed. The morphological changes of neutrophil nuclei clearly indicated to us that ANCA-induced NETs were of nuclear rather than of mitochondrial origin, as recently desc...

There is emerging evidence that platelets are major contributors to inflammatory processes through intimate associations with innate immune cells. Here, we report that activated platelets induce the formation of neutrophil extracellular traps (NETs) in transfusion-related acute lung injury (TRALI), which is the leading cause of death after transfusion therapy. NETs are composed of decondensed chromatin decorated with granular proteins that function to trap extracellular pathogens; their formation requires the activation of neutrophils and release of their DNA in a process that may or may not result in neutrophil death. In a mouse model of TRALI that is neutrophil and platelet dependent, NETs appeared in the lung microvasculature and NET components increased in the plasma. We detected NETs in the lungs and plasma of human TRALI and in the plasma of patients with acute lung injury. In the experimental TRALI model, targeting platelet activation with either aspirin or a glycoprotein IIb/IIIa inhibitor decreased NET formation and lung injury. We then directly targeted NET components with a histone blocking antibody and DNase1, both of which protected mice from TRALI. These data suggest that NETs contribute to lung endothelial injury and that targeting NET formation may be a promising new direction for the treatment of acute lung injury.

Neutrophils, the most abundant type of leukocytes in blood, can form neutrophil extracellular traps (NETs). These are pathogen-trapping structures generated by expulsion of the neutrophil's DNA with associated proteolytic enzymes. NETs produced by infection can promote cancer metastasis. Here, we show that metastatic breast cancer cells can induce neutrophils to form metastasis-supporting NETs in the absence of infection. Using intravital imaging, we observed NET-like structures around metastatic 4T1 cancer cells that had reached the lungs of mice. We also found NETs in clinical samples of triple-negative human breast cancer. The formation of NETs stimulated the invasion and migration of breast cancer cells in vitro. Inhibiting NET formation or digesting NETs with DNase I blocked these processes. Treatment with NET-digesting, DNase I-coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs by cancer cells is a previously unidentified metastasis-promoting tumor-host interaction and a potential therapeutic target.

Despite major advances in understanding the molecular and genetic basis of cancer, metastasis remains the cause of >90% of cancer-related mortality1. Understanding metastasis initiation and progression is critical to developing new therapeutic strategies to treat and prevent metastatic disease. Prevailing theories hypothesize that metastases are seeded by rare tumour cells with unique properties, which may function like stem cells in their ability to initiate and propagate metastatic tumours2–5. However, the identity of metastasis-initiating cells in human breast cancer remains elusive, and whether metastases are hierarchically organized is unknown2. Here we show at the single-cell level that early stage metastatic cells possess a distinct stem-like gene expression signature. To identify and isolate metastatic cells from patient-derived xenograft models of human breast cancer, we developed a highly sensitive fluorescence-activated cell sorting (FACS)-based assay, which allowed us to enumerate metastatic cells in mouse peripheral tissues. We compared gene signatures in metastatic cells from tissues with low versus high metastatic burden. Metastatic cells from low-burden tissues were distinct owing to their increased expression of stem cell, epithelial-to-mesenchymal transition, pro-survival, and dormancy-associated genes. By contrast, metastatic cells from high-burden tissues were similar to primary tumour cells, which were more heterogeneous and expressed higher levels of luminal differentiation genes. Transplantation of stem-like metastatic cells from low-burden tissues showed that they have considerable tumour-initiating capacity, and can differentiate to produce luminal-like cancer cells. Progression to high metastatic burden was associated with increased proliferation and MYC expression, which could be attenuated by treatment with cyclin-dependent kinase (CDK) inhibitors. These findings support a hierarchical model for metastasis, in which metastases are initiated by stem-like cells that proliferate and differentiate to produce advanced metastatic disease.

Breast cancer arises from breast epithelial cells that acquire genetic alterations leading to subsequent loss of tissue homeostasis. Several distinct epithelial subpopulations have been proposed, but complete understanding of the spectrum of heterogeneity and differentiation hierarchy in the human breast remains elusive. Here, we use single-cell mRNA sequencing (scRNAseq) to profile the transcriptomes of 25,790 primary human breast epithelial cells isolated from reduction mammoplasties of seven individuals. Unbiased clustering analysis reveals the existence of three distinct epithelial cell populations, one basal and two luminal cell types, which we identify as secretory L1- and hormone-responsive L2-type cells. Pseudotemporal reconstruction of differentiation trajectories produces one continuous lineage hierarchy that closely connects the basal lineage to the two differentiated luminal branches. Our comprehensive cell atlas provides insights into the cellular blueprint of the human breast epithelium and will form the foundation to understand how the system goes awry during breast cancer.

During wound healing in adult mouse skin, hair follicles and then adipocytes regenerate. Adipocytes regenerate from myofibroblasts, a specialized contractile wound fibroblast. Here we study wound fibroblast diversity using single-cell RNA-sequencing. On analysis, wound fibroblasts group into twelve clusters. Pseudotime and RNA velocity analyses reveal that some clusters likely represent consecutive differentiation states toward a contractile phenotype, while others appear to represent distinct fibroblast lineages. One subset of fibroblasts expresses hematopoietic markers, suggesting their myeloid origin. We validate this finding using single-cell western blot and single-cell RNA-sequencing on genetically labeled myofibroblasts. Using bone marrow transplantation and Cre recombinase-based lineage tracing experiments, we rule out cell fusion events and confirm that hematopoietic lineage cells give rise to a subset of myofibroblasts and rare regenerated adipocytes. In conclusion, our study reveals that wounding induces a high degree of heterogeneity among fibroblasts and recruits highly plastic myeloid cells that contribute to adipocyte regeneration.