Macrophages Maintain Epithelium Integrity by Limiting Fungal Product Absorption.

The colon is primarily responsible for absorbing fluids. It contains a large number of microorganisms including fungi, which are enriched in its distal segment. The colonic mucosa must therefore tightly regulate fluid influx to control absorption of fungal metabolites, which can be toxic to epithelial cells and lead to barrier dysfunction. How this is achieved remains unknown. Here, we describe a mechanism by which the innate immune system allows rapid quality check of absorbed fluids to avoid intoxication of colonocytes. This mechanism relies on a population of distal colon macrophages that are equipped with "balloon-like" protrusions (BLPs) inserted in the epithelium, which sample absorbed fluids. In the absence of macrophages or BLPs, epithelial cells keep absorbing fluids containing fungal products, leading to their death and subsequent loss of epithelial barrier integrity. These results reveal an unexpected and essential role of macrophages in the maintenance of colon-microbiota interactions in homeostasis. VIDEO ABSTRACT.

Epithelial colonization by gut dendritic cells promotes their functional diversification.

Dendritic cells (DCs) patrol tissues and transport antigens to lymph nodes to initiate adaptive immune responses. Within tissues, DCs constitute a complex cell population composed of distinct subsets that can exhibit different activation states and functions. How tissue-specific cues orchestrate DC diversification remains elusive. Here, we show that the small intestine included two pools of cDC2s originating from common pre-DC precursors: (1) lamina propria (LP) CD103+CD11b+ cDC2s that were mature-like proinflammatory cells and (2) intraepithelial cDC2s that exhibited an immature-like phenotype as well as tolerogenic properties. These phenotypes resulted from the action of food-derived retinoic acid (ATRA), which enhanced actomyosin contractility and promoted LP cDC2 transmigration into the epithelium. There, cDC2s were imprinted by environmental cues, including ATRA itself and the mucus component Muc2. Hence, by reaching distinct subtissular niches, DCs can exist as immature and mature cells within the same tissue, revealing an additional mechanism of DC functional diversification.

The emerging roles of the cytoskeleton in intestinal epithelium homeostasis.

The intestinal epithelium must absorb many nutrients and water while forming a barrier that is impermeable to pathogens present in the external environment. Concurrently to fulfill this dual role, the intestinal epithelium is challenged by a rapid renewal of cells and forces resulting from digestion. Hence, intestinal homeostasis requires precise control of tissue integrity, tissue renewal, cell polarity, and force generation/transmission. In this review, we highlight the contribution of the cell cytoskeleton- actin, microtubules, and intermediate filaments- to intestinal epithelium homeostasis. With a focus on enterocytes, we first discuss the role of these networks in the formation and maintenance of cell-cell and cell-matrix junctions. Then, we cover their role in intracellular trafficking related to the apicobasal polarity of enterocytes. Finally, we report on the cytoskeletal changes that occur during tissue renewal. In conclusion, the importance of the cytoskeleton in maintaining intestinal homeostasis is emerging, and we think this field will keep evolving.

Gut homeostasis at a glance.

The intestine, a rapidly self-renewing organ, is part of the gastrointestinal system. Its major roles are to absorb food-derived nutrients and water, process waste and act as a barrier against potentially harmful substances. Here, we will give a brief overview of the primary functions of the intestine, its structure and the luminal gradients along its length. We will discuss the dynamics of the intestinal epithelium, its turnover, and the maintenance of homeostasis. Finally, we will focus on the characteristics and functions of intestinal mesenchymal and immune cells. In this Cell Science at a Glance article and the accompanying poster, we aim to present the most recent information about gut cell biology and physiology, providing a resource for further exploration.

Endothelial cell invasion is controlled by dactylopodia.

Sprouting angiogenesis is fundamental for development and contributes to cancer, diabetic retinopathy, and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on how tip cells invade into tissues. Here, we show that endothelial tip cells use dactylopodia as the main cellular protrusion for invasion into nonvascular extracellular matrix. We show that dactylopodia and filopodia protrusions are balanced by myosin IIA (NMIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of NMIIA promotes excessive dactylopodia formation in detriment of filopodia. Conversely, endothelial cell-autonomous ablation of Arp2/3 prevents dactylopodia development and leads to excessive filopodia formation. We further show that NMIIA inhibits Rac1-dependent activation of Arp2/3 by regulating the maturation state of focal adhesions. Our discoveries establish a comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way toward strategies to block invasive tip cells during sprouting angiogenesis.

Self-generated gradients steer collective migration on viscoelastic collagen networks.

Growing evidence suggests that the physical properties of the cellular microenvironment influence cell migration. However, it is not currently understood how active physical remodelling by cells affects migration dynamics. Here we report that cell clusters seeded on deformable collagen-I networks display persistent collective migration despite not showing any apparent intrinsic polarity. Clusters generate transient gradients in collagen density and alignment due to viscoelastic relaxation of the collagen networks. Combining theory and experiments, we show that crosslinking collagen networks or reducing cell cluster size results in reduced network deformation, shorter viscoelastic relaxation time and smaller gradients, leading to lower migration persistence. Traction force and Brillouin microscopy reveal asymmetries in force distributions and collagen stiffness during migration, providing evidence of mechanical cross-talk between cells and their substrate during migration. This physical model provides a mechanism for self-generated directional migration on viscoelastic substrates in the absence of internal biochemical polarity cues.

Cancer cells in the tumor core exhibit spatially coordinated migration patterns.

In the early stages of metastasis, cancer cells exit the primary tumor and enter the vasculature. Although most studies have focused on the tumor invasive front, cancer cells from the tumor core can also potentially metastasize. To address cell motility in the tumor core, we imaged tumor explants from spontaneously forming tumors in mice in real time using long-term two-photon microscopy. Cancer cells in the tumor core are remarkably dynamic and exhibit correlated migration patterns, giving rise to local 'currents' and large-scale tissue dynamics. Although cells exhibit stop-and-start migration with intermittent pauses, pausing does not appear to be required during division. Use of pharmacological inhibitors indicates that migration patterns in tumors are actively driven by the actin cytoskeleton. Under these conditions, we also observed a relationship between migration speed and correlation length, suggesting that cells in tumors are near a jamming transition. Our study provides new insight into the dynamics of cancer cells in the tumor core, opening new avenues of research in understanding the migratory properties of cancer cells and later metastasis.This article has an associated First Person interview with the first author of the paper.

Reentrant wetting transition in the spreading of cellular aggregates.

We study spreading on soft substrates of cellular aggregates using CT26 cells that produce an extracellular matrix (ECM). Compared to our previous work on the spreading of S180 cellular aggregates, which did not secrete ECMs, we found that the spreading velocity of the precursor film is also maximal for intermediate rigidities, but new striking features show up. First, we observed a cascade of liquid-gas-liquid (L/G/L) transitions of the precursor film as the substrate rigidity is decreased. We attribute the L/G transition to a decrease of cell/cell adhesion resulting from the weakening of the cell/substrate adhesion. We attribute the reentrant liquid phase (G/L) observed on soft substrates to the slow spreading of the aggregates on ultra-soft substrates, which gives time to the cells to secrete more ECM proteins and stick together. Second, a nematic order appears in the cohesive (liquid) states of the precursor film, attributed to the gradient of cell's velocities.

Enterocyte loss of polarity and gut wound healing rely upon the F-actin-severing function of villin.

Efficient wound healing is required to maintain the integrity of the intestinal epithelial barrier because of its constant exposure to a large variety of environmental stresses. This process implies a partial cell depolarization and the acquisition of a motile phenotype that involves rearrangements of the actin cytoskeleton. Here we address how polarized enterocytes harboring actin-rich apical microvilli undergo extensive cell remodeling to drive injury repair. Using live imaging technologies, we demonstrate that enterocytes in vitro and in vivo rapidly depolarize their microvilli at the wound edge. Through its F-actin-severing activity, the microvillar actin-binding protein villin drives both apical microvilli disassembly in vitro and in vivo and promotes lamellipodial extension. Photoactivation experiments indicate that microvillar actin is mobilized at the lamellipodium, allowing optimal migration. Finally, efficient repair of colonic mechanical injuries requires villin severing of F-actin, emphasizing the importance of villin function in intestinal homeostasis. Thus, villin severs F-actin to ensure microvillus depolarization and enterocyte remodeling upon injury. This work highlights the importance of specialized apical pole disassembly for the repolarization of epithelial cells initiating migration.

Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro.

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.

Compressive stress inhibits proliferation in tumor spheroids through a volume limitation.

In most instances, the growth of solid tumors occurs in constrained environments and requires a competition for space. A mechanical crosstalk can arise from this competition. In this article, we dissect the biomechanical sequence caused by a controlled compressive stress on multicellular spheroids (MCSs) used as a tumor model system. On timescales of minutes, we show that a compressive stress causes a reduction of the MCS volume, linked to a reduction of the cell volume in the core of the MCS. On timescales of hours, we observe a reversible induction of the proliferation inhibitor, p27Kip1, from the center to the periphery of the spheroid. On timescales of days, we observe that cells are blocked in the cell cycle at the late G1 checkpoint, the restriction point. We show that the effect of pressure on the proliferation can be antagonized by silencing p27Kip1. Finally, we quantify a clear correlation between the pressure-induced volume change and the growth rate of the spheroid. The compression-induced proliferation arrest that we studied is conserved for five cell lines, and is completely reversible. It demonstrates a generic crosstalk between mechanical stresses and the key players of cell cycle regulation. Our results suggest a role of volume change in the sensitivity to pressure, and that p27Kip1 is strongly influenced by this change.

EGF/EGFR axis contributes to the progression of cholangiocarcinoma through the induction of an epithelial-mesenchymal transition.

BACKGROUND & AIMS: Epithelial-mesenchymal transition (EMT) is a cellular process involved in cancer progression. The first step of EMT consists in the disruption of E-cadherin-mediated adherens junctions. Cholangiocarcinoma (CCA), a cancer with a poor prognosis due to local invasion and metastasis, displays EMT features. EGFR, a receptor tyrosine kinase, plays a major role in CCA progression. The aim of the study was to determine if EMT is induced by EGFR in CCA cells. METHODS: In vivo, the expression of E-cadherin was analysed in CCA tumours of 100 patients and correlated with pathological features and EGFR expression, and in a xenograft model in mice treated with gefitinib, an inhibitor of EGFR. In vitro, the regulation of EMT by EGFR was investigated in CCA cell lines. RESULTS: In human CCA, a cytoplasmic localization of E-cadherin occurred in 50% of the tumours was associated with the peripheral type of CCA, tumour size, the presence of satellite nodules and EGFR overexpression. In xenografted tumours, E-cadherin displayed a cytoplasmic pattern whereas the treatment of mice with gefitinib restored the membranous expression of E-cadherin. In vitro, EGF induced scattering of CCA cells that resulted from the disruption of adherens junctions. Internalization and decreased expression of E-cadherin, as well as nuclear translocation of ß-catenin, were observed in EGF-treated CCA cells. In these cells, EMT-transcription factors (i.e., Slug and Zeb-1) and mesenchymal markers (i.e., N-cadherin and α-SMA) were induced, favoring cell invasiveness through cytoskeleton remodeling. All these effects were inhibited by gefitinib. CONCLUSIONS: The EGF/EGFR axis triggers EMT in CCA cells highlighting the key role of this pathway in CCA progression.

Mechanical forces across compartments coordinate cell shape and fate transitions to generate tissue architecture.

Morphogenesis and cell state transitions must be coordinated in time and space to produce a functional tissue. An excellent paradigm to understand the coupling of these processes is mammalian hair follicle development, which is initiated by the formation of an epithelial invagination-termed placode-that coincides with the emergence of a designated hair follicle stem cell population. The mechanisms directing the deformation of the epithelium, cell state transitions and physical compartmentalization of the placode are unknown. Here we identify a key role for coordinated mechanical forces stemming from contractile, proliferative and proteolytic activities across the epithelial and mesenchymal compartments in generating the placode structure. A ring of fibroblast cells gradually wraps around the placode cells to generate centripetal contractile forces, which, in collaboration with polarized epithelial myosin activity, promote elongation and local tissue thickening. These mechanical stresses further enhance compartmentalization of Sox9 expression to promote stem cell positioning. Subsequently, proteolytic remodelling locally softens the basement membrane to facilitate a release of pressure on the placode, enabling localized cell divisions, tissue fluidification and epithelial invagination into the underlying mesenchyme. Together, our experiments and modelling identify dynamic cell shape transformations and tissue-scale mechanical cooperation as key factors for orchestrating organ formation.

Compressive stress triggers fibroblasts spreading over cancer cells to generate carcinoma in situ organization.

At the early stage of tumor progression, fibroblasts are located at the outer edges of the tumor, forming an encasing layer around it. In this work, we have developed a 3D in vitro model where fibroblasts' layout resembles the structure seen in carcinoma in situ. We use a microfluidic encapsulation technology to co-culture fibroblasts and cancer cells within hollow, permeable, and elastic alginate shells. We find that in the absence of spatial constraint, fibroblasts and cancer cells do not mix but segregate into distinct aggregates composed of individual cell types. However, upon confinement, fibroblasts enwrap cancer cell spheroid. Using a combination of biophysical methods and live imaging, we find that buildup of compressive stress is required to induce fibroblasts spreading over the aggregates of tumor cells. We propose that compressive stress generated by the tumor growth might be a mechanism that prompts fibroblasts to form a capsule around the tumor.

Mechanical control of cell flow in multicellular spheroids.

Collective cell motion is observed in a wide range of biological processes. In tumors, physiological gradients of nutrients, growth factors, or even oxygen give rise to gradients of proliferation. We show using fluorescently labeled particles that these gradients drive a velocity field resulting in a cellular flow in multicellular spheroids. Under mechanical stress, the cellular flow is drastically reduced. We describe the results with a hydrodynamic model that considers only convection of the particles by the cellular flow.

Optimized protocol for the generation of an orthotopic colon cancer mouse model and metastasis.

The microenvironment plays an essential role in tumor development and metastatic progression. Here, we describe a simple and rapid protocol to generate tumors in mice using colon cancer cell lines or tumoroids in the correct microenvironment, colonic mucosa. We also detail steps for monitoring the growth of the primary tumor in real time using colonoscopy or in vivo imaging system, as well as monitoring metastasis development. Finally, we describe tissue collection and sample preparation for subsequent immunohistochemistry analysis.

Fibroblasts generate topographical cues that steer cancer cell migration.

Fibroblasts play a fundamental role in tumor development. Among other functions, they regulate cancer cells' migration through rearranging the extracellular matrix, secreting soluble factors, and establishing direct physical contacts with cancer cells. Here, we report that migrating fibroblasts deposit on the substrate a network of tubular structures that serves as a guidance cue for cancer cell migration. Such membranous tubular network, hereafter called tracks, is stably anchored to the substrate in a ß5-integrin-dependent manner. We found that cancer cells specifically adhere to tracks by using clathrin-coated structures that pinch and engulf tracks. Tracks thus represent a spatial memory of fibroblast migration paths that is read and erased by cancer cells directionally migrating along them. We propose that fibroblast tracks represent a topography-based intercellular communication system capable of steering cancer cell migration.

Correction: Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions.

Correction for 'Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions' by Marine Verhulsel et al., Lab Chip, 2021, 21, 365-377, https://doi.org/10.1039/d0lc00672f.

Cancer-associated fibroblasts actively compress cancer cells and modulate mechanotransduction.

During tumor progression, cancer-associated fibroblasts (CAFs) accumulate in tumors and produce an excessive extracellular matrix (ECM), forming a capsule that enwraps cancer cells. This capsule acts as a barrier that restricts tumor growth leading to the buildup of intratumoral pressure. Combining genetic and physical manipulations in vivo with microfabrication and force measurements in vitro, we found that the CAFs capsule is not a passive barrier but instead actively compresses cancer cells using actomyosin contractility. Abrogation of CAFs contractility in vivo leads to the dissipation of compressive forces and impairment of capsule formation. By mapping CAF force patterns in 3D, we show that compression is a CAF-intrinsic property independent of cancer cell growth. Supracellular coordination of CAFs is achieved through fibronectin cables that serve as scaffolds allowing force transmission. Cancer cells mechanosense CAF compression, resulting in an altered localization of the transcriptional regulator YAP and a decrease in proliferation. Our study unveils that the contractile capsule actively compresses cancer cells, modulates their mechanical signaling, and reorganizes tumor morphology.