Cytoplasmic accumulation and plasma membrane association of anillin and Ect2 promote confined migration and invasion.

Cells migrating in confinement experience mechanical challenges whose consequences on cell migration machinery remain only partially understood. Here, we demonstrate that a pool of the cytokinesis regulatory protein anillin is retained during interphase in the cytoplasm of different cell types. Confinement induces recruitment of cytoplasmic anillin to plasma membrane at the poles of migrating cells, which is further enhanced upon nuclear envelope (NE) rupture(s). Rupture events also enable the cytoplasmic egress of predominantly nuclear RhoGEF Ect2. Anillin and Ect2 redistributions scale with microenvironmental stiffness and confinement, and are observed in confined cells in vitro and in invading tumor cells in vivo. Anillin, which binds actomyosin at the cell poles, and Ect2, which activates RhoA, cooperate additively to promote myosin II contractility, and promote efficient invasion and extravasation. Overall, our work provides a mechanistic understanding of how cytokinesis regulators mediate RhoA/ROCK/myosin II-dependent mechanoadaptation during confined migration and invasive cancer progression.

Cytokinesis machinery promotes cell dissociation from collectively migrating strands in confinement.

Cells tune adherens junction dynamics to regulate epithelial integrity in diverse (patho)physiological processes, including cancer metastasis. We hypothesized that the spatially confining architecture of peritumor stroma promotes metastatic cell dissemination by remodeling cell-cell adhesive interactions. By combining microfluidics with live-cell imaging, FLIM/FRET biosensors, and optogenetic tools, we show that confinement induces leader cell dissociation from cohesive ensembles. Cell dissociation is triggered by myosin IIA (MIIA) dismantling of E-cadherin cell-cell junctions, as recapitulated by a mathematical model. Elevated MIIA contractility is controlled by RhoA/ROCK activation, which requires distinct guanine nucleotide exchange factors (GEFs). Confinement activates RhoA via nucleocytoplasmic shuttling of the cytokinesis-regulatory proteins RacGAP1 and Ect2 and increased microtubule dynamics, which results in the release of active GEF-H1. Thus, confining microenvironments are sufficient to induce cell dissemination from primary tumors by remodeling E-cadherin cell junctions via the interplay of microtubules, nuclear trafficking, and RhoA/ROCK/MIIA pathway and not by down-regulating E-cadherin expression.

Plasticity of cancer invasion and energy metabolism.

Energy deprivation is a frequent adverse event in tumors that is caused by mutations, malperfusion, hypoxia, and nutrition deficit. The resulting bioenergetic stress leads to signaling and metabolic adaptation responses in tumor cells, secures survival, and adjusts migration activity. The kinetic responses of cancer cells to energy deficit were recently identified, including a switch of invasive cancer cells to energy-conservative amoeboid migration and an enhanced capability for distant metastasis. We review the energy programs employed by different cancer invasion modes including collective, mesenchymal, and amoeboid migration, as well as their interconversion in response to energy deprivation, and we discuss the consequences for metastatic escape. Understanding the energy requirements of amoeboid and other dissemination strategies offers rationales for improving therapeutic targeting of metastatic cancer progression.

Dissecting the recruitment and self-organization of αSMA-positive fibroblasts in the foreign body response.

The foreign body response (FBR) is a clinically relevant issue that can cause malfunction of implanted medical devices by fibrotic encapsulation. Whereas inflammatory aspects of the FBR have been established, underlying fibroblast-dependent mechanisms remain unclear. We here combine multiphoton microscopy with ad hoc reporter mice expressing α-smooth muscle actin (αSMA) protein to determine the locoregional fibroblast dynamics, activation, and fibrotic encapsulation of polymeric materials. Fibroblasts invaded as individual cells and established a multicellular network, which transited to a two-compartment fibrotic response displaying an αSMA cold external capsule and a long-lasting, inner αSMA hot environment. The recruitment of fibroblasts and extent of fibrosis were only incompletely inhibited after depletion of macrophages, implicating coexistence of macrophage-dependent and macrophage-independent mediators. Furthermore, neither altering material type or porosity modulated αSMA+ cell recruitment and distribution. This identifies fibroblast activation and network formation toward a two-compartment FBR as a conserved, self-organizing process partially independent of macrophages.

223Ra Induces Transient Functional Bone Marrow Toxicity.

223Ra is a bone-seeking, α-particle-emitting radionuclide approved for the treatment of patients with metastatic prostate cancer and is currently being tested in a variety of clinical trials for primary and metastatic cancers to bone. Clinical evaluation of 223Ra hematologic safety showed a significantly increased rate of neutropenia and thrombocytopenia in patients, hinting at myelosuppression as a side effect. Methods: In this study, we investigated the consequences of 223Ra treatment on bone marrow biology by combining flow cytometry, single-cell RNA sequencing, three-dimensional multiphoton microscopy and bone marrow transplantation analyses. Results: 223Ra accumulated in bones and induced zonal radiation damage confined to the bone interface, followed by replacement of the impaired areas with adipocyte infiltration, as monitored by 3-dimensional multiphoton microscopy ex vivo. Flow cytometry and single-cell transcriptomic analyses on bone marrow hematopoietic populations revealed transient, nonspecific 223Ra-mediated cytotoxicity on resident populations, including stem, progenitor, and mature leukocytes. This toxicity was paralleled by a significant decrease in white blood cells and platelets in peripheral blood-an effect that was overcome within 40 d after treatment. 223Ra exposure did not impair full hematopoietic reconstitution, suggesting that bone marrow function is not permanently hampered. Conclusion: Our results provide a comprehensive explanation of 223Ra reversible effects on bone marrow cells and exclude long-term myelotoxicity, supporting safety for patients.

Deep Learning for Automated Analysis of Cellular and Extracellular Components of the Foreign Body Response in Multiphoton Microscopy Images.

The Foreign body response (FBR) is a major unresolved challenge that compromises medical implant integration and function by inflammation and fibrotic encapsulation. Mice implanted with polymeric scaffolds coupled to intravital non-linear multiphoton microscopy acquisition enable multiparametric, longitudinal investigation of the FBR evolution and interference strategies. However, follow-up analyses based on visual localization and manual segmentation are extremely time-consuming, subject to human error, and do not allow for automated parameter extraction. We developed an integrated computational pipeline based on an innovative and versatile variant of the U-Net neural network to segment and quantify cellular and extracellular structures of interest, which is maintained across different objectives without impairing accuracy. This software for automatically detecting the elements of the FBR shows promise to unravel the complexity of this pathophysiological process.

Dorsoventral polarity directs cell responses to migration track geometries.

How migrating cells differentially adapt and respond to extracellular track geometries remains unknown. Using intravital imaging, we demonstrate that invading cells exhibit dorsoventral (top-to-bottom) polarity in vivo. To investigate the impact of dorsoventral polarity on cell locomotion through different confining geometries, we fabricated microchannels of fixed cross-sectional area, albeit with distinct aspect ratios. Vertical confinement, exerted along the dorsoventral polarity axis, induces myosin II-dependent nuclear stiffening, which results in RhoA hyperactivation at the cell poles and slow bleb-based migration. In lateral confinement, directed perpendicularly to the dorsoventral polarity axis, the absence of perinuclear myosin II fails to increase nuclear stiffness. Hence, cells maintain basal RhoA activity and display faster mesenchymal migration. In summary, by integrating microfabrication, imaging techniques, and intravital microscopy, we demonstrate that dorsoventral polarity, observed in vivo and in vitro, directs cell responses in confinement by spatially tuning RhoA activity, which controls bleb-based versus mesenchymal migration.

Molecular detection of Pseudomonas aeruginosa in recreational water.

The aim of this study was the development of a new molecular assay for Pseudomonas aeruginosa identification in recreational water. The method includes bacterial cell concentration through membrane filtration, a short (6 h) culture-enrichment step, DNA extraction and its amplification through a Real-Time PCR assay. The performance of the molecular approach was evaluated on 44 samples of swimming pool water and compared with the reference method UNI EN ISO 16266:2008. Positivity rates of 6% and 74% in pool and inlet water, respectively, with the standard culture method, and of 23% and 74% with the molecular method were found. Statistical analysis indicated "substantial agreement" (Cohen's Kappa index: 0.6831) between the two approaches. RAPD typing of P. aeruginosa isolates showed identical fingerprint profiles, indicating their epidemiological correlation. The developed protocol showed very high specificity and a detection limit of 10 genomic units. This technique has the potential to screen large numbers of environmental samples, and could be proposed as part of a self-monitoring plan for recreational facilities, improving surveillance and early warning systems.