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.

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.

A catalog of numerical centrosome defects in epithelial ovarian cancers.

Centrosome amplification, the presence of more than two centrosomes in a cell is a common feature of most human cancer cell lines. However, little is known about centrosome numbers in human cancers and whether amplification or other numerical aberrations are frequently present. To address this question, we have analyzed a large cohort of primary human epithelial ovarian cancers (EOCs) from 100 patients. We found that rigorous quantitation of centrosome number in tumor samples was extremely challenging due to tumor heterogeneity and extensive tissue disorganization. Interestingly, even if centrosome clusters could be identified, the incidence of centrosome amplification was not comparable to what has been described in cultured cancer cells. Surprisingly, centrosome loss events where a few or many nuclei were not associated with centrosomes were clearly noticed and overall more frequent than centrosome amplification. Our findings highlight the difficulty of characterizing centrosome numbers in human tumors, while revealing a novel paradigm of centrosome number defects in EOCs.

Stand by me: Fibroblasts regulation of the intestinal epithelium during development and homeostasis.

The epithelium of the small intestine is composed of a single layer of cells that line two functionally distinct compartments, the villi that project into the lumen of the gut and the crypts that descend into the underlying connective tissue. Stem cells are located in crypts, where they divide and give rise to transit-amplifying cells that differentiate into secretory and absorptive epithelial cells. Most differentiated cells travel upwards from the crypt towards the villus tip, where they shed into the lumen. While some of these cell behaviors are an intrinsic property of the epithelium, it is becoming evident that tight coordination between the epithelium and the underlying fibroblasts plays a critical role in tissue morphogenesis, stem-cell niche maintenance and regionalized gene expression along the crypt-villus axis. Here, we will review the current literature describing the interaction between epithelium and fibroblasts during crypt-villus axis development and intestinal epithelium renewal during homeostasis.

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.

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.

Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration.

Intestinal organoids capture essential features of the intestinal epithelium such as crypt folding, cellular compartmentalization and collective movements. Each of these processes and their coordination require patterned forces that are at present unknown. Here we map three-dimensional cellular forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the extracellular matrix and folds through apical constriction, whereas the transit amplifying zone pulls the extracellular matrix and elongates through basal constriction. The size of the stem cell compartment depends on the extracellular-matrix stiffness and endogenous cellular forces. Computational modelling reveals that crypt shape and force distribution rely on cell surface tensions following cortical actomyosin density. Finally, cells are pulled out of the crypt along a gradient of increasing tension. Our study unveils how patterned forces enable compartmentalization, folding and collective migration in the intestinal epithelium.

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.

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

Organoids are widely used as a model system to study gut pathophysiology; however, they fail to fully reproduce the complex, multi-component structure of the intestinal wall. We present here a new gut on chip model that allows the co-culture of primary epithelial and stromal cells. The device has the topography and dimensions of the mouse gut and is based on a 3D collagen I scaffold. The scaffold is coated with a thin layer of laminin to mimic the basement membrane. To maintain the scaffold structure while preserving its cytocompatibility, the collagen scaffold was rigidified by threose-based post-polymerization treatment. This treatment being cytocompatible enabled the incorporation of primary intestinal fibroblasts inside the scaffold, reproducing the gut stromal compartment. We observed that mouse organoids, when deposited into crypts, opened up and epithelialized the scaffold, generating a polarized epithelial monolayer. Proper segregation of dividing and differentiated cells along the crypt-villus axis was achieved under these conditions. Finally, we show that the application of fluid shear stress allows the long-term culture of this intestinal epithelium. Our device represents a new biomimetic tool that captures key features of the gut complexity and could be used to study gut pathophysiology.

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.

A new biomimetic assay reveals the temporal role of matrix stiffening in cancer cell invasion.

Tumor initiation and growth is associated with significant changes in the surrounding tissue. During carcinoma progression, a global stiffening of the extracellular matrix is observed and is interpreted as a signature of aggressive invasive tumors. However, it is still unknown whether this increase in matrix rigidity promotes invasion and whether this effect is constant along the course of invasion. Here we have developed a biomimetic in vitro assay that enabled us to address the question of the importance of tissue rigidity in the chronology of tumor invasion. Using low concentrations of the sugar threose, we can effectively stiffen reconstituted collagen I matrices and control the stiffening in time with no direct effect on residing cells. Our findings demonstrate that, depending on the timing of its stiffening, the extracellular matrix could either inhibit or promote cancer cell invasion and subsequent metastasis: while matrix stiffening after the onset of invasion promotes cancer cell migration and tumor spreading, stiff matrices encapsulate the tumor at an early stage and prevent cancer cell invasion. Our study suggests that adding a temporal dimension in in vitro models to analyze biological processes in four dimensions is necessary to fully capture their complexity.

3D cell migration in the presence of chemical gradients using microfluidics.

Chemotaxis is an important biological process involved in the development of multicellular organisms, immune response and cancer metastasis. In order to better understand how cells follow chemical cues in their native environments, we recently developed a microfluidics-based chemotaxis device that allows for observation of cells or cell aggregates in 3D networks in response to tunable chemical gradients (Aizel et al., 2017). Here, we describe the methods required for fabrication of this device as well as its use for live imaging experiments and subsequent analysis of imaging data. This device can be adapted to study a number of different cell arrangements and chemical gradients, opening new avenues of research in 3D chemotaxis.

Frustrated endocytosis controls contractility-independent mechanotransduction at clathrin-coated structures.

It is generally assumed that cells interrogate the mechanical properties of their environment by pushing and pulling on the extracellular matrix (ECM). For instance, acto-myosin-dependent contraction forces exerted at focal adhesions (FAs) allow the cell to actively probe substrate elasticity. Here, we report that a subset of long-lived and flat clathrin-coated structures (CCSs), also termed plaques, are contractility-independent mechanosensitive signaling platforms. We observed that plaques assemble in response to increasing substrate rigidity and that this is independent of FAs, actin and myosin-II activity. We show that plaque assembly depends on αvß5 integrin, and is a consequence of frustrated endocytosis whereby αvß5 tightly engaged with the stiff substrate locally stalls CCS dynamics. We also report that plaques serve as platforms for receptor-dependent signaling and are required for increased Erk activation and cell proliferation on stiff environments. We conclude that CCSs are mechanotransduction structures that sense substrate rigidity independently of cell contractility.

Author Correction: Cancer-associated fibroblasts induce metalloprotease-independent cancer cell invasion of the basement membrane.

In the original version of this Article, financial support and contributions in manuscript preparation were not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include the following:'M.P. and P.O. would like to thank Prof. Roderick Y.H. Lim for advice during manuscript preparation and for providing the laboratory and microscopy infrastructure.… [We also thank] the NanoteraProject, awarded to the PATLiSciII Consortium (M.P and P.O)…'.

Cell Migration in Tissues: Explant Culture and Live Imaging.

Cell migration is a process that ensures correct cell localization and function in development and homeostasis. In disease such as cancer, cells acquire an upregulated migratory capacity that leads to their dissemination throughout the body. Live imaging of cell migration allows for better understanding of cell behaviors in development, adult tissue homeostasis and disease. We have optimized live imaging procedures to track cell migration in adult murine tissue explants derived from: (1) healthy gut; (2) primary intestinal carcinoma; and (3) the liver, a common metastatic site. To track epithelial cell migration in the gut, we generated an inducible fluorescent reporter mouse, enabling us to visualize and track individual cells in unperturbed gut epithelium. To image intratumoral cancer cells, we use a spontaneous intestinal cancer model based on the activation of Notch1 and deletion of p53 in the mouse intestinal epithelium, which gives rise to aggressive carcinoma. Interaction of cancer cells with a metastatic niche, the mouse liver, is addressed using a liver colonization model. In summary, we describe a method for long-term 3D imaging of tissue explants by two-photon excitation microscopy. Explant culturing and imaging can help understand dynamic behavior of cells in homeostasis and disease, and would be applicable to various tissues.

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.

Cancer-associated fibroblasts induce metalloprotease-independent cancer cell invasion of the basement membrane.

At the stage of carcinoma in situ, the basement membrane (BM) segregates tumor cells from the stroma. This barrier must be breached to allow dissemination of the tumor cells to adjacent tissues. Cancer cells can perforate the BM using proteolysis; however, whether stromal cells play a role in this process remains unknown. Here we show that an abundant stromal cell population, cancer-associated fibroblasts (CAFs), promote cancer cell invasion through the BM. CAFs facilitate the breaching of the BM in a matrix metalloproteinase-independent manner. Instead, CAFs pull, stretch, and soften the BM leading to the formation of gaps through which cancer cells can migrate. By exerting contractile forces, CAFs alter the organization and the physical properties of the BM, making it permissive for cancer cell invasion. Blocking the ability of stromal cells to exert mechanical forces on the BM could therefore represent a new therapeutic strategy against aggressive tumors.Stromal cells play various roles in tumor establishment and metastasis. Here the authors, using an ex-vivo model, show that cancer-associated fibroblasts facilitate colon cancer cells invasion in a matrix metalloproteinase-independent manner, likely by pulling and stretching the basement membrane to form gaps.

A tuneable microfluidic system for long duration chemotaxis experiments in a 3D collagen matrix.

In many cell types, migration can be oriented towards a chemical stimulus. In mammals, for example, embryonic cells migrate to follow developmental cues, immune cells migrate toward sites of inflammation, and cancer cells migrate away from the primary tumour and toward blood vessels during metastasis. Understanding how cells migrate in 3D environments in response to chemical cues is thus crucial to understanding directed migration in normal and disease states. To date, chemotaxis in mammalian cells has been primarily studied using 2D migration models. However, it is becoming increasingly clear that the mechanisms by which cells migrate in 2D and 3D environments dramatically differ, and cells in their native environments are confronted with a complex chemical milieu. To address these issues, we developed a microfluidic device to monitor the behaviour of cells embedded in a 3D collagen matrix in the presence of complex concentration fields of chemoattractants. This tuneable microsystem enables the generation of (1) homogeneous, stationary gradients set by a purely diffusive mechanism, or (2) spatially evolving, stationary gradients, set by a convection-diffusion mechanism. The device allows for stable gradients over several days and is large enough to study the behaviour of large cell aggregates. We observe that primary mature dendritic cells respond uniformly to homogeneous diffusion gradients, while cell behaviour is highly position-dependent in spatially variable convection-diffusion gradients. In addition, we demonstrate a directed response of cancer cells migrating away from tumour-like aggregates in the presence of soluble chemokine gradients. Together, this microfluidic device is a powerful system to observe the response of different cells and aggregates to tuneable chemical gradients.