Biology subjects

Cambi, A.

Publications and source records attributed to Cambi, A..

6 recordsLinked to original sources

bioRxiv2024

In vitro-generated inflammatory fibroblasts secrete extracellular matrix with biochemical and biophysical properties similar to tissue-remodelling fibroblasts

In solid cancers, inflammation and viral infections, two main fibroblast subtypes have been identified: myofibroblast-like fibroblasts and inflammatory fibroblasts. In the tumour microenvironment (TME), these cancer-associated fibroblast (CAF) subtypes are known as myCAFs, which generate a stiffened fibrotic extracellular matrix (ECM), and iCAFs, which secrete inflammatory cytokines to locally modulate the immune response. Yet, whether iCAFs contribute to shaping the ECM biochemical and biophysical properties remains unknown, mainly because robust in vitro models to generate fibroblast subtypes are lacking. Here, we established an in vitro cell culture system based on murine NIH3T3 fibroblasts and stimulation by TGF{beta} and IL1, alone or in combination, to induce fibroblast subtypes. Gene expression analysis of well-documented myCAF (Acta2/Tagln) and iCAF (Ccl2/Il6/Lif) markers revealed that TGF{beta} induced a myCAF-like phenotype, while a combination of TGF{beta} and IL1 induced an iCAF-like phenotype. We compared these in vitro subtypes to myCAFs and iCAFs from publicly available scRNAseq data of tumour tissues from cancer patients. We found that, similar to myCAFs, both tumour-associated and in vitro iCAFs express Acta2/Tagln as well as genes encoding for typical ECM proteins, which correlated in vitro with the ability to contract collagen. Furthermore, fluorescence microscopy and atomic force microscopy revealed that in vitro both subtypes generate thick, layered and stiff matrices with highly aligned ECM, demonstrating for the first time that iCAFs may also contribute to a pathological ECM. Finally, matrices generated from these in vitro fibroblast subtypes, but not from uninduced or IL1-only stimulated fibroblasts, enhanced the expression of the immune suppression marker Arg1 in co-cultured macrophages. Our study provides new insights in the contribution of inflammatory fibroblasts to ECM deposition and remodelling and puts forward a well-defined in vitro model to generate different fibroblast subtypes for future in-depth mechanistic studies of their roles in cancer and other pathologies.

cell biology↗

bioRxiv2021

Chemo-mechanical Diffusion Waves Orchestrate Collective Dynamics of Immune Cell Podosomes

Immune cells, such as macrophages and dendritic cells, can utilize podosomes, actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. In these cells, individual podosomes exhibit periodic protrusion and retraction cycles (vertical oscillations) to probe their microenvironment, while multiple podosomes arranged in clusters demonstrate coordinated wave-like spatiotemporal dynamics. However, the mechanisms governing both the individual vertical oscillations and the coordinated oscillation waves in clusters remain unclear. By integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for both the oscillatory growth of individual podosomes and wave-like dynamics in clusters. Our model reveals that podosomes show oscillatory growth when the actin polymerization-associated protrusion and the signaling-associated myosin contraction occur at similar rates, while the diffusion of actin monomers within the cluster drives mesoscale coordination of individual podosome oscillations in an apparent wave-like fashion. Our model predicts the influence of different pharmacological treatments targeting myosin activity, actin polymerization, and mechanosensitive pathways, as well as the impact of the microenvironment stiffness on the wavelengths, frequencies, and speeds of the chemo-mechanical waves. Overall, our integrated theoretical and experimental approach reveals how collective wave dynamics arise due to the coupling between chemo-mechanical signaling and actin diffusion, shedding light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy.

biophysics↗

bioRxiv2020

Characterization of the signalling modalities of prostaglandin E2 receptors EP2 and EP4 reveals crosstalk and a role for microtubules

Prostaglandin E2 (PGE2) is a lipid mediator that modulates the function of myeloid immune cells such as macrophages and dendritic cells (DCs) through the activation of the G protein-coupled receptors EP2 and EP4. While both EP2 and EP4 signalling leads to an elevation of intracellular cyclic adenosine monophosphate (cAMP) levels through the stimulating Gs protein, EP4 also couples to the inhibitory Gi protein to decrease the production of cAMP. The receptor-specific contributions to downstream immune modulatory functions are still poorly defined. Here, we employed quantitative imaging methods to characterize the early EP2 and EP4 signalling events in myeloid cells and their contribution to the dissolution of adhesion structures called podosomes, which is a first and essential step in DC maturation. We first show that podosome loss in DCs is primarily mediated by EP4. Next, we demonstrate that EP2 and EP4 signalling leads to distinct cAMP production profiles, with EP4 inducing a transient cAMP response and EP2 inducing a sustained cAMP response only at high PGE2 levels. We further find that simultaneous EP2 and EP4 stimulation attenuates cAMP production, suggesting a reciprocal control of EP2 and EP4 signaling. Finally, we demonstrate that efficient signaling of both EP2 and EP4 relies on an intact microtubule network. Together, these results enhance our understanding of early EP2 and EP4 signalling in myeloid cells. Considering that modulation of PGE2 signalling is regarded as an important therapeutic possibility in anti-tumour immunotherapy, our findings may facilitate the development of efficient and specific immune modulators of PGE2 receptors.

immunology↗

bioRxiv2020

Membrane receptor MerTK is a newly identified transcriptional regulator that associates to chromatin as nanoclusters during human DC differentiation

MerTK is a transmembrane receptor tyrosine kinase (RTK) mainly expressed in dendritic cells (DCs) and macrophages where it plays an important role in immunotolerance, but also in activating oncogenic signalling pathways. Albeit MerTK is exploited as clinical target in cancer and auto-immune disorders, the mechanisms that regulate its diverse functions are poorly understood. Here, we identified a remarkably high pool of the full receptor in the nucleus of human DCs. Nuclear translocation was ligand-dependent. Importantly, MerTK nuclear levels correlated to DC differentiation and were spatiotemporally regulated by the transmembrane receptor LRP-1. Using dual-colour super-resolution nanoscopy we discovered that nuclear MerTK forms nanoclusters, whose strength strongly depends on chromatin accessibility during DC differentiation. We finally revealed high transcription capacity of MerTK. Overall, our work indicates that nuclear MerTK acts as a transcription factor regulating DC differentiation, thus implicating for the first time a physiological function for RTK nuclear translocation in immunity.

immunology↗

bioRxiv2019

Intracellular Galectin-9 controls dendritic cell function by maintaining plasma membrane rigidity

Extracellular Galectins constitute a novel mechanism of membrane protein organisation at the cell surface. Although Galectins are also highly expressed intracellularly, their cytosolic functions are poorly understood. Here, we investigated the role of Galectin-9 in dendritic cell (DC) surface organisation and function. By combining functional, super-resolution and atomic force microscopy experiments to analyse membrane stiffness, we identified intracellular Galectin-9 to be indispensable for plasma membrane integrity and structure in DCs. Galectin-9 knockdown studies revealed intracellular Galectin-9 to directly control cortical membrane structure via modulating Rac1 activity, providing the underlying mechanism of Galectin-9-dependent actin cytoskeleton organisation. Consequent to its role in maintaining plasma membrane structure, phagocytosis studies revealed that Galectin-9 was essential for C-type lectin receptor-mediated pathogen uptake by human DCs. This was confirmed by the impaired phagocytic capacity of Galectin-9-null murine DCs. Together, this study demonstrates a novel role for intracellular Galectin-9 in modulating DC function, which may be evolutionary conserved.

cell biology↗

bioRxiv2019

Modular actin nano-architecture enables podosome protrusion and mechanosensing

Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and podosomes. An adequate structural framework for this process is still missing. Here, we reveal the modular actin nano-architecture that enables podosome protrusion and mechanosensing. The podosome protrusive core contains a central branched actin module encased by a linear actin module, each harboring specific actin interactors and actin isoforms. From the core, two actin modules radiate: ventral filaments bound by vinculin and connected to the plasma membrane and dorsal interpodosomal filaments crosslinked by myosin IIA. On stiff substrates, the actin modules mediate long-range substrate exploration, associated with degradative behavior. On compliant substrates, the vinculin-bound ventral actin filaments shorten, resulting in short-range connectivity and a focally protrusive, non-degradative state. Our findings redefine podosome nanoscale architecture and reveal a paradigm for how actin modularity drives invadosome mechanosensing in cells that breach tissue boundaries.

cell biology↗