Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the ...encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell–cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution.
During development, epithelial tissues undergo extensive morphogenesis based on coordinated changes of cell shape and position over time. Continuum mechanics describes tissue mechanical state and ...shape changes in terms of strain and stress. It accounts for individual cell properties using only a few spatially averaged material parameters. To determine the mechanical state and parameters in the Drosophila pupa dorsal thorax epithelium, we severed in vivo the adherens junctions around a disc-shaped domain comprising typically a hundred cells. This enabled a direct measurement of the strain along different orientations at once. The amplitude and the anisotropy of the strain increased during development. We also measured the stress-to-viscosity ratio and similarly found an increase in amplitude and anisotropy. The relaxation time was of the order of 10 s. We propose a space–time, continuous model of the relaxation. Good agreement with experimental data validates the description of the epithelial domain as a continuous, linear, visco-elastic material. We discuss the relevant time and length scales. Another material parameter, the ratio of external friction to internal viscosity, is estimated by fitting the initial velocity profile. Together, our results contribute to quantify forces and displacements, and their time evolution, during morphogenesis.
Epithelial-to-mesenchymal transition-like (EMT-like) is a critical process allowing initiation of metastases during tumour progression. Here, to investigate its role in intestinal cancer, we combine ...computational network-based and experimental approaches to create a mouse model with high metastatic potential. Construction and analysis of this network map depicting molecular mechanisms of EMT regulation based on the literature suggests that Notch activation and p53 deletion have a synergistic effect in activating EMT-like processes. To confirm this prediction, we generate transgenic mice by conditionally activating the Notch1 receptor and deleting p53 in the digestive epithelium (NICD/p53(-/-)). These mice develop metastatic tumours with high penetrance. Using GFP lineage tracing, we identify single malignant cells with mesenchymal features in primary and metastatic tumours in vivo. The development of such a model that recapitulates the cellular features observed in invasive human colorectal tumours is appealing for innovative drug discovery.
Abstract Chemotherapy enhances the antitumor adaptive immune T cell response, but the immunosuppressive tumor environment often dominates, resulting in cancer relapse. Antigen-presenting cells such ...as tumor-associated macrophages (TAMs) and tumor dendritic cells (TuDCs) are the main protagonists of tumor-infiltrating lymphocyte (TIL) immuno-suppression. TAMs have been widely investigated and are associated with poor prognosis, but the immuno-suppressive activity of TuDCs is less well understood. We performed two-photon imaging of the tumor tissue to examine the spatiotemporal interactions between TILs and TuDCs after chemotherapy. In a strongly immuno-suppressive murine tumor model, cyclophosphamide-mediated chemotherapy transiently enhanced the antitumor activity of adoptively transferred ovalbumin-specific CD8+ T cell receptor transgenic T cells (OTI) but barely affected TuDC compartment within the tumor. Time lapse imaging of living tumor tissue showed that TuDCs are organized as a mesh with dynamic interconnections. Once infiltrated into the tumor parenchyma, OTI T cells make antigen-specific and long-lasting contacts with TuDCs. Extensive analysis of TIL infiltration on histologic section revealed that after chemotherapy the majority of OTI T cells interact with TuDCs and that infiltration is restricted to TuDC-rich areas. We propose that the TuDC network exerts antigen-dependent unproductive retention that trap T cells and limit their antitumor effectiveness.
Complete activation of B cells relies on their capacity to extract tethered antigens from immune synapses by either exerting mechanical forces or promoting their proteolytic degradation through ...lysosome secretion. Whether antigen extraction can also be tuned by local cues originating from the lymphoid microenvironment has not been investigated. We here show that the expression of Galectin-8—a glycan-binding protein found in the extracellular milieu, which regulates interactions between cells and matrix proteins—is increased within lymph nodes under inflammatory conditions where it enhances B cell arrest phases upon antigen recognition in vivo and promotes synapse formation during BCR recognition of immobilized antigens. Galectin-8 triggers a faster recruitment and secretion of lysosomes toward the B cell-antigen contact site, resulting in efficient extraction of immobilized antigens through a proteolytic mechanism. Thus, extracellular cues can determine how B cells sense and extract tethered antigens and thereby tune B cell responses in vivo.
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•Galectin-8 reinforces B cell arrest phases upon antigen recognition in vivo•Galectin-8 sustains BCR signaling during recognition of immobilized antigens•This enhances lysosome secretion and favors the proteolytic extraction of antigens•Galectin-8 improves the capacity of B cells to present antigens to helper T cells
Obino et al. report that Galectin-8 interacts with the BCR, promotes B cell arrest phases during surface-tethered antigen encounter, and facilitates synapse formation and lysosome secretion, which favors the proteolytic extraction of antigens. Consequently, Galectin-8 increases the capacity of B cells to present antigens to helper T cells in vivo.
During metastasis, cancer cells breach the basement membrane and migrate through the stroma mostly composed of a network of collagen I fibers. Cell migration on 2D is initiated by protrusion of the ...cell membrane followed by formation of adhesions that link the actin cytoskeleton to the extracellular matrix (ECM). Cells then move forwards by exerting traction forces on the adhesions at its front and by disassembling adhesions at the rear. In 2D, only the ventral surface of a migrating cell is in contact with the ECM, where cell–matrix adhesions are assembled. In 3D matrices, even though the whole surface of a migrating cell is available for interacting with the ECM, it is unclear whether discrete adhesion structures actually exist. Using high-resolution confocal microscopy we imaged the endogenous adhesome proteins in three different cancer cell types embedded in non-pepsinized collagen type I, polymerized at a slow rate, to allow the formation of a network that resembles the organization of EMC observed in vivo. Vinculin aggregates were detected in the cellular protrusions, frequently colocalizing with collagen fibers, implying they correspond to adhesion structures in 3D. As the distance from the substrate bottom increases, adhesion aggregates become smaller and almost undetectable in some cell lines. Using intravital imaging we show here, for the first time, the existence of adhesome proteins aggregates in vivo. These aggregates share similarities with the ones found in 3D collagen matrices. It still remains to be determined if adhesions assembled in 3D and in vivo share functional similarities to the well-described adhesions in 2D. This will provide a major step forward in understanding cell migration in more physiological environments.
Many signaling and metabolic pathways rely on the ability of some of the proteins involved to undergo a substrate-induced transition between at least two structural states. Among the various models ...put forward to account for binding and activity curves of those allosteric proteins, the Monod, Wyman, and Changeux model for allostery theory has certainly been the most influential, although a central postulate, the preexisting equilibrium between the low-activity, low-affinity quaternary structure and the high-activity, high-affinity quaternary structure states in the absence of substrates, has long awaited direct experimental substantiation. Upon substrate binding, allosteric Escherichia coli aspartate transcarbamoylase adopts alternate quaternary structures, stabilized by a set of interdomain and intersubunit interactions, which are readily differentiated by their solution x-ray scattering curves. Disruption of a salt link, which is observed only in the low-activity, low-affinity quaternary structure, between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain yields a mutant enzyme that is in a reversible equilibrium between at least two states in the absence of ligand, a major tenet of the Monod, Wyman, and Changeux model. By using this mutant as a magnifying glass of the structural effect of ligand binding, a comparative analysis of the binding of carbamoyl phosphate (CP) and analogs points out the crucial role of the amine group of CP in facilitating the transition toward the high-activity, high-affinity quaternary state. Thus, the cooperative binding of aspartate in aspartate transcarbamoylase appears to result from the combination of the preexisting quaternary structure equilibrium with local changes induced by CP binding.
The response of cells to forces is essential for tissue morphogenesis and homeostasis. This response has been extensively investigated in interphase cells, but it remains unclear how forces affect ...dividing cells. We used a combination of micro-manipulation tools on human dividing cells to address the role of physical parameters of the micro-environment in controlling the cell division axis, a key element of tissue morphogenesis. We found that forces applied on the cell body direct spindle orientation during mitosis. We further show that external constraints induce a polarization of dynamic subcortical actin structures that correlate with spindle movements. We propose that cells divide according to cues provided by their mechanical micro-environment, aligning daughter cells with the external force field.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Although regulatory T cells (T regs ) are known to suppress self-reactive autoimmune responses, their role during T cell responses to nonself antigens is not well understood. We show that T regs play ...a critical role during the priming of immune responses in mice. T reg depletion induced the activation and expansion of a population of low-avidity CD8⁺ T cells because of overproduction of CCL-3/4/5 chemokines, which stabilized the interactions between antigen-presenting dendritic cells and low-avidity T cells. In the absence of T regs , the avidity of the primary immune response was impaired, which resulted in reduced memory to Listeria monocytogenes. These results suggest that T regs are important regulators of the homeostasis of CD8⁺ T cell priming and play a critical role in the induction of high-avidity primary responses and effective memory.
The initiation of cytotoxic immune responses requires the direct interaction between naive CD8+ T lymphocytes and dendritic cells (DCs). Multiphoton imaging in intact lymph nodes (LNs) showed that ...during priming, naive T cells and DCs establish sequentially brief (i.e., minutes) and long (hours) antigen-specific contacts. We show here that the expression of the Intercellular Adhesion Molecule-1 (ICAM-1) by mature DCs is critical for long-lasting contacts with CD8+ T cells but dispensable for short-lived antigen-specific interactions. Serial brief DC-T cell contacts induced early CD8+ T cell activation, proliferation, and differentiation into effector cytotoxic T lymphocytes in the first few days after immunization. ICAM-1-deficient mature DCs, however, failed to induce fully effective priming, because CD8+ T cells produced reduced amounts of interferon γ and were clonally depleted after 2 weeks. In addition, Icam1−/− mice failed to respond to rechallenge. We conclude that ICAM-1-dependent long-lasting interactions between mature DCs and naive CD8+ T cells determine the survival of activated CD8+ T cells and the establishment of effective memory.
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