Cell Crowding Research Articles

Growth of tumours with stem cells: The effect of crowding and ageing of cells

Mathematical models for the growth of tumours in the presence of stem cells (CSCs) and differentiated tumour cells (CCs) are presented and discussed. The CSCs are assumed to be immortal and multipotent, i.e. capable of generating several possible lineages of CCs that may undergo ageing and apoptosis. Each CC is characterised by two indexes, related to the differentiation lineage and the class of age, respectively. Furthermore, the effect of crowding is taken into account, assuming that mitosis can be hindered by the presence of cells in the vicinity of the would-be mother cell. Two families of models are proposed. First, models based on cellular automata are considered, whose evolution is governed by stochastic rules. Then, by averaging over the cells with the same pair of indexes, we obtain a deterministic model that consists of a system of Ordinary Differential Equations (ODEs) whose unknown functions are the fractions of the cells in each lineage and the class of age. The system presents a basic novelty with respect to the other compartmental models proposed in the literature as it cannot be solved hierarchically because of the presence of the crowding effect. Numerical simulations based on the two families of models give the same qualitative results and, in particular, they evidentiate the occurrence of the tumour paradox: an increased mortality of the CCs may induce a faster growth of the tumour. A final section of the paper is devoted to the case in which the age distribution of the CCs is continuous and not discrete. In this case, an interesting mathematical problem is obtained that consists of one ODE for the fraction of CSCs and m first-order Partial Differential Equations (PDEs); one for each lineage of CCs.

Single-cell resolved imaging reveals intra-tumor heterogeneity in glycolysis, transitions between metabolic states, and their regulatory mechanisms

SummaryInter-cellular heterogeneity in metabolic state has been proposed to influence many cancer phenotypes, including responses to targeted therapy. Here, we track the transitions and heritability of metabolic states in single PIK3CA mutant breast cancer cells, identify non-genetic glycolytic heterogeneity, and build on observations derived from methods reliant on bulk analyses. Using fluorescent biosensors in vitro and in tumors, we have identified distinct subpopulations of cells whose glycolytic and mitochondrial metabolism are regulated by combinations of phosphatidylinositol 3-kinase (PI3K) signaling, bromodomain activity, and cell crowding effects. The actin severing protein cofilin, as well as PI3K, regulates rapid changes in glucose metabolism, whereas treatment with the bromodomain inhibitor slowly abrogates a subpopulation of cells whose glycolytic activity is PI3K independent. We show how bromodomain function and PI3K signaling, along with actin remodeling, independently modulate glycolysis and how targeting these pathways affects distinct subpopulations of cancer cells.

Conventional Direct Smear Yields Diagnostic Indicators of Gastric-Type Mucinous Carcinoma Compared with Cytomorphological Features Identified by Liquid-Based Cervical Cytology

Introduction: Gastric-type mucinous carcinoma (GAS) of the uterine cervix is an adenocarcinoma subtype with a gastric phenotype that poses diagnostic pitfalls in cervical screening cytology because of its blunt morphologic atypia and the limited utility of human papillomavirus testing and ancillary immunochemical staining. Despite the recent widespread uptake of liquid-based cytology (LBC) systems, the cytomorphological features of GAS in LBC samples and the differential features between GAS and usual-type endocervical adenocarcinoma (UEA) remain unclear. Methods: Eight GAS cases, all of which were surgically treated following histological confirmation, were examined. Direct Papanicolaou-stained smears and LBC samples were reviewed and compared with 10 UEA cases as controls. Featured cytomorphological findings were as follows: background (mucinous, inflammatory, or necrotic), cell crowding (size of neoplastic cell clusters), cytoplasm (golden mucin and cell border), and nuclei (nuclear chromatin and nucleoli). Results: Of 18 adenocarcinomas, 16 were detected against a non-mucinous background in LBC samples, most of which were accompanied by mild to moderate inflammation. Clusters comprising >300 neoplastic cells were identified in both GAS and UEA in conventional smears (CSs), while no LBC samples harboured clusters as large as these. Cell borders of GAS were more distinct than those of UEA in CSs (p < 0.001), although fewer populations of neoplastic clusters revealed distinct cell borders in both GAS and UEA in LBC samples. Three of 8 and 2 of 8 GAS cases had golden mucin in CSs and in LBC samples, respectively, which was not detected in UEA at all. Nucleoli against fine nuclear chromatin were more pronounced in GAS than in UEA on CS (p = 0.03), although the difference between GAS and UEA was not apparent in LBC samples. Discussion/Conclusion: This study demonstrated that the diagnostic clues to detect GAS using the conventional approach, namely distinct cell borders and prominent nucleoli, are not useful for excluding UEA in LBC samples. Conventional cervical smears may indicate a diagnosis of GAS; however, specific high-risk HPV detection approaches, such as HPV test or immunocytochemical p16/Ki-67 dual staining, are desirable to differentiate GAS from UEA in the setting of LBC with ambiguous cytomorphological features.

3D-printable cell crowding device enables imaging of live cells in compression.

We designed and fabricated, using low-cost 3D printing technologies, a device that enables direct control of cell density in epithelial monolayers. The device operates by varying the tension of a silicone substrate upon which the cells are adhered. Multiple devices can be manufactured easily and placed in any standard incubator. This allows long-term culturing of cells on pretensioned substrates until the user decreases the tension, thereby inducing compressive forces in plane and subsequent instantaneous cell crowding. Moreover, the low-profile device is completely portable and can be mounted directly onto an inverted optical microscope. This enables visualization of the morphology and dynamics of living cells in stretched or compressed conditions using a wide range of high-resolution microscopy techniques.

Coarse-grained modeling of cell division in 3D: influence of density, medium viscosity, and inter-membrane friction on cell growth and nearest neighbor distribution

ABSTRACT Considerable experimental and theoretical research has been dedicated to understanding the connection between the biochemical activity of cells and their mechanical environment. This is exemplified by the common structures of developing epithelial cells between various species and the decay of cell population growth rate over time. We study these two phenomena in a system of simulated cells with identical mechanical properties, and growth factors in both epithelial and 3D configurations embedded in viscous fluid. We demonstrate that the increase in the density of cellular systems and the consequential crowding of cells by their neighbors are crucial factors in causing the decay of tissue growth rate. We also show that tissue structure can be reproduced by a purely mechanical model with a more faithful treatment of inter-membrane interactions. Finally, we also show that, assuming all other factors constant, growth, and final structure depends on inter-membrane and medium friction coefficients. Of these two, the latter has a stronger influence on slowing down growth and disrupting structure.

Blocking Cell Extrusion Prevents Bronchoconstriction-Induced Airway Epithelial Damage and Inflammation

Asthma is a common disease characterized by airway constriction, excess mucus, and inflammation. We previously showed that cell crowding drives steady-state epithelial cell extrusion and cell death. Using ex vivo lung slices, we show that excessive crowding from methacholine-induced bronchoconstriction causes extensive cell extrusion leading to destruction of the airway epithelial barrier. In live mice, bronchoconstriction-induced extrusion caused airway epithelia damage and inflammation in secondary and tertiary airways. In primary airways, bronchoconstriction triggered mucus secretion by a mechanism reminiscent of extrusion. Reversing bronchoconstriction with albuterol failed to ameliorate these symptoms, whereas inhibiting the stretch-activated calcium channel, Piezo1, prevented extrusion, inflammation, and mucus secretion. Our findings propose a new etiology for asthma where the mechanical crowding from bronchoconstriction causes so much extrusion that it compromises the barrier, triggering subsequent inflammation. Furthermore, inhibiting extrusion, potentially with gadolinium, an inexpensive Piezo1 inhibitor, may prevent the asthma inflammatory cycle.

Live imaging of ERK signalling dynamics in differentiating mouse embryonic stem cells.

Stimulation of the ERK/MAPK pathway is required for the exit from pluripotency and onset of differentiation in mouse embryonic stem cells (ESCs). The dynamic behaviour of ERK activity in individual cells during this transition is unclear. Using a FRET-based biosensor, we monitored ERK signalling dynamics of single mouse ESCs during differentiation. ERK activity was highly heterogeneous, with considerable variability in ERK signalling between single cells within ESC colonies. Different triggers of differentiation induced distinct ERK activity profiles. Surprisingly, the dynamic features of ERK signalling were not strongly coupled to loss of pluripotency marker expression, regardless of the differentiation stimulus, suggesting the normal dynamic range of ERK signalling is not rate-limiting in single cells during differentiation. ERK signalling dynamics were sensitive to the degree of cell crowding and were similar in neighbouring cells. Sister cells from a mitotic division also showed more similar ERK activity, an effect that was apparent whether cells remained adjacent or moved apart after division. These data suggest a combination of cell lineage and niche contributes to the absolute level of ERK signalling in mouse ESCs.

Fluctuations in cell density alter protein markers of multiple cellular compartments, confounding experimental outcomes.

The life cycle of cultured proliferating cells is characterized by fluctuations in cell population density induced by periodic subculturing. This leads to corresponding changes in micro- and macroenvironment of the cells, accompanied by altered cellular metabolism, growth rate and locomotion. Studying cell density-dependent morphological, physiological and biochemical fluctuations is relevant for understanding basic cellular mechanisms and for uncovering the intrinsic variation of commonly used tissue culture experimental models. Using multiple cell lines, we found that expression levels of the autophagic markers p62 and LC3II, and lysosomal enzyme cathepsin D were altered in highly confluent cells as a consequence of nutrient depletion and cell crowding, which led to inactivation of the mTOR signaling pathway. Furthermore, both Lamp1 and active focal adhesion kinase (FAK) were reduced in high-density cells, while chemical inhibition or deletion of FAK led to alterations in lysosomal and autophagic proteins, as well as in the mTOR signaling. This was accompanied by alterations in the Hippo signaling pathway, while cell cycle checkpoint regulator p-cdc2 remained unaffected in at least one studied cell line. On the other hand, allometric scaling of cellular compartments in growing cell populations resulted in biochemically detectable changes in the plasma membrane proteins Na+K+-ATPase and cadherin, and nuclear proteins HDAC1 and Lamin B1. Finally, we demonstrate how treatment-induced changes in cell density and corresponding modulation of susceptible proteins may lead to ambiguous experimental outcomes, or erroneous interpretation of cell culture data. Together, our data emphasize the need to recognize cell density as an important experimental variable in order to improve scientific rigor of cell culture-based studies.

Spatial confinement downsizes the inflammatory response of macrophages.

Macrophages respond to chemical/metabolic and physical stimuli, but their effects cannot be readily decoupled in vivo during pro-inflammatory activation. Here, we show that preventing macrophage spreading by spatial confinement, as imposed by micropatterning, microporous substrates or cell crowding, suppresses late lipopolysaccharide (LPS)-activated transcriptional programs (biomarkers IL-6, CXCL9, IL-1β, and iNOS) by mechanomodulating chromatin compaction and epigenetic alterations (HDAC3 levels and H3K36-dimethylation). Mechanistically, confinement reduces actin polymerization, thereby lowers the LPS-stimulated nuclear translocation of MRTF-A. This lowers the activity of the MRTF-A-SRF complex and subsequently downregulates the inflammatory response, as confirmed by chromatin immunoprecipitation coupled with quantitative PCR and RNA sequencing analysis. Confinement thus downregulates pro-inflammatory cytokine secretion and, well before any activation processes, the phagocytic potential of macrophages. Contrarily, early events, including activation of the LPS receptor TLR4, and downstream NF-κB and IRF3 signalling and hence the expression of early LPS-responsive genes were marginally affected by confinement. These findings have broad implications in the context of mechanobiology, inflammation and immunology, as well as in tissue engineering and regenerative medicine.

Osteoblasts infill irregular pores under curvature and porosity controls: a hypothesis-testing analysis of cell behaviours.

The geometric control of bone tissue growth plays a significant role in bone remodelling, age-related bone loss, and tissue engineering. However, how exactly geometry influences the behaviour of bone-forming cells remains elusive. Geometry modulates cell populations collectively through the evolving space available to the cells, but it may also modulate the individual behaviours of cells. To factor out the collective influence of geometry and gain access to the geometric regulation of individual cell behaviours, we develop a mathematical model of the infilling of cortical bone pores and use it with available experimental data on cortical infilling rates. Testing different possible modes of geometric controls of individual cell behaviours consistent with the experimental data, we find that efficient smoothing of irregular pores only occurs when cell secretory rate is controlled by porosity rather than curvature. This porosity control suggests the convergence of a large scale of intercellular signalling to single bone-forming cells, consistent with that provided by the osteocyte network in response to mechanical stimulus. After validating the mathematical model with the histological record of a real cortical pore infilling, we explore the infilling of a population of randomly generated initial pore shapes. We find that amongst all the geometric regulations considered, the collective influence of curvature on cell crowding is a dominant factor for how fast cortical bone pores infill, and we suggest that the irregularity of cement lines thereby explains some of the variability in double labelling data as well as the overall speed of osteon infilling.

ПНЕВМОНЭКТОМИЯ У ПАЦИЕНТА С ОСЛОЖНЕННЫМ ТЕЧЕНИЕМ ГРАНУЛЕМАТОЗА ВЕГЕНЕРА

The article describes a clinical case of 42-year old male patient with necrotising granulomatosis with polyangitis (Wegener's granulomatosis) complicated by pulmonary aspergillosis, and due to the latter a life-saving pneumonectomy had to be done Microbiological tests of surgery specimens detected the cell crowding of mold fungi of Aspergillus fumigatus The histological tests showed necrotising granulomatosis with polyangitis, formation of cavities with fibrotic walls and fungal population; fibrinous-hemorrhagic pleurisy. In one month after surgery the patient was transferred to the rheumatological ward, where biological anti-B-cellular therapy with rituximab was started with a consequent positive outcome.

Dynamics of 3D carcinoma cell invasion into aligned collagen.

Carcinoma cells frequently expand and invade from a confined lesion, or multicellular clusters, into and through the stroma on the path to metastasis, often with an efficiency dictated by the architecture and composition of the microenvironment. Specifically, in desmoplastic carcinomas such as those of the breast, aligned collagen tracks provide contact guidance cues for directed cancer cell invasion. Yet, the evolving dynamics of this process of invasion remains poorly understood, in part due to difficulties in continuously capturing both spatial and temporal heterogeneity and progression to invasion in experimental systems. Therefore, to study the local invasion process from cell dense clusters into aligned collagen architectures found in solid tumors, we developed a novel engineered 3D invasion platform that integrates an aligned collagen matrix with a cell dense tumor-like plug. Using multiphoton microscopy and quantitative analysis of cell motility, we track the invasion of cancer cells from cell-dense bulk clusters into the pre-aligned 3D matrix, and define the temporal evolution of the advancing invasion fronts over several days. This enables us to identify and probe cell dynamics in key regions of interest: behind, at, and beyond the edge of the invading lesion at distinct time points. Analysis of single cell migration identifies significant spatial heterogeneity in migration behavior between cells in the highly cell-dense region behind the leading edge of the invasion front and cells at and beyond the leading edge. Moreover, temporal variations in motility and directionality are also observed between cells within the cell-dense tumor-like plug and the leading invasive edge as its boundary extends into the anisotropic collagen over time. Furthermore, experimental results combined with mathematical modeling demonstrate that in addition to contact guidance, physical crowding of cells is a key regulating factor orchestrating variability in single cell migration during invasion into anisotropic ECM. Thus, our novel platform enables us to capture spatio-temporal dynamics of cell behavior behind, at, and beyond the invasive front and reveals heterogeneous, local interactions that lead to the emergence and maintenance of the advancing front.

High-throughput Microscopic Analysis of Salmonella Invasion of Host Cells.

Salmonella is a Gram-negative bacterium causing a gastro-enteric disease called salmonellosis. During the first phase of infection, Salmonella uses its flagella to swim near the surface of the epithelial cells and to target specific site of infection. In order to study the selection criteria that determine which host cells are targeted by the pathogen, and to analyze the relation between infecting Salmonella (i.e., cooperation or competition), we have established a high-throughput microscopic assay of HeLa cells sequentially infected with fluorescent bacteria. Using an automated pipeline of image analysis, we quantitatively characterized a multitude of parameters of infected and non-infected cells. Based on this, we established a predictive model that allowed us to identify those parameters involved in host cell vulnerability towards infection. We revealed that host cell vulnerability has two origins: a pathogen-induced cellular vulnerability emerging from Salmonella uptake and persisting at later stages of the infection process; and a host cell-inherent vulnerability linked with cell inherent attributes, such as local cell crowding, and cholesterol content. Our method forecasts the probability of Salmonella infection within monolayers of epithelial cells based on morphological or molecular host cell parameters. Here, we provide a detailed description of the workflow including the computer-based analysis pipeline. Our method has the potential to be applied to study other combinations of host-pathogen interactions.

Identification of Parameters of Host Cell Vulnerability during Salmonella Infection by Quantitative Image Analysis and Modeling.

Salmonella targets and enters epithelial cells at permissive entry sites: some cells are more likely to be infected than others. However, the parameters that lead to host cell heterogeneity are not known. Here, we quantitatively characterized host cell vulnerability to Salmonella infection based on imaged parameters. We performed successive infections of the same host cell population followed by automated high-throughput microscopy and observed that infected cells have a higher probability of being reinfected. Establishing a predictive model, we identified two combined origins of host cell vulnerability: pathogen-induced cellular vulnerability emerging from Salmonella uptake and persisting at later stages of the infection and host cell-inherent vulnerability. We linked the host cell-inherent vulnerability with its morphological attributes, such as local cell crowding, and with host cell cholesterol content. This showed that the probability of Salmonella infection success can be forecast from morphological or molecular host cell parameters.

The ubiquitin ligase ITCH coordinates small intestinal epithelial homeostasis by modulating cell proliferation, differentiation, and migration

Maintenance of the intestinal mucosa is driven by local signals that coordinate epithelial proliferation, differentiation, and turnover in order to separate antigenic luminal contents from the host's immune system. Breaches in this barrier promote gastrointestinal pathologies ranging from inflammatory bowel disease to cancer. The ubiquitin ligase ITCH is known to regulate immune responses, and loss of function of ITCH has been associated with gastrointestinal inflammatory disorders, particularly in the colon. However, the small intestine appears to be spared from this pathology. Here we explored the physiological mechanism that underlies the preservation of mucosal homeostasis in the small intestine in mice lacking ITCH (Itcha18H/a18H). Histological analysis of the small intestines from young adult mice revealed architectural changes in animals deficient for ITCH, including villus blunting with cell crowding, crypt expansion, and thickening of the muscularis propria relative to age-matched mice sufficient for ITCH. These differences were more prominent in the distal part of the small intestine and were not dependent upon lymphoid cells. Underlying the observed changes in the epithelium were expansion of the Ki67+ proliferating transit amplifying progenitor population and increased numbers of terminally differentiated mucus-secreting goblet and anti-microbial producing Paneth cells, which are both important in controlling local inflammation in the small intestine and are known to be dysregulated in inflammatory bowel disease. Homeostasis in the small intestine of Itcha18H/a18H animals was maintained by increased cell turnover, including accelerated migration of epithelial cells along the crypt-villus axis and increased apoptosis of epithelial cells at the crypt-villus junction. Consistent with this enhanced turnover, Itcha18H/a18H mice carrying the Min mutation (Itcha18H/a18H; ApcMin/+) displayed a 76% reduction in tumor burden as compared to ApcMin/+ littermates with normal levels of ITCH. These findings highlight the role of ITCH as an important modulator of intestinal epithelial homeostasis.

TMEFF2 shedding is regulated by oxidative stress and mediated by ADAMs and transmembrane serine proteases implicated in prostate cancer.

TMEFF2 is a type I transmembrane protein with two follistatin (FS) and one EGF‐like domain over‐expressed in prostate cancer; however its biological role in prostate cancer development and progression remains unclear, which may, at least in part, be explained by its proteolytic processing. The extracellular part of TMEFF2 (TMEFF2‐ECD) is cleaved by ADAM17 and the membrane‐retained fragment is further processed by the gamma‐secretase complex. TMEFF2 shedding is increased with cell crowding, a condition associated with the tumour microenvironment, which was mediated by oxidative stress signalling, requiring jun‐kinase (JNK) activation. Moreover, we have identified that TMEFF2 is also a novel substrate for other proteases implicated in prostate cancer, including two ADAMs (ADAM9 and ADAM12) and the type II transmembrane serine proteinases (TTSPs) matriptase‐1 and hepsin. Whereas cleavage by ADAM9 and ADAM12 generates previously identified TMEFF2‐ECD, proteolytic processing by matriptase‐1 and hepsin produced TMEFF2 fragments, composed of TMEFF2‐ECD or FS and/or EGF‐like domains as well as novel membrane retained fragments. Differential TMEFF2 processing from a single transmembrane protein may be a general mechanism to modulate transmembrane protein levels and domains, dependent on the repertoire of ADAMs or TTSPs expressed by the target cell.

Intestinal Stem Cell Pool Regulation in Drosophila.

SummaryIntestinal epithelial renewal is mediated by intestinal stem cells (ISCs) that exist in a state of neutral drift, wherein individual ISC lineages are regularly lost and born but ISC numbers remain constant. To test whether an active mechanism maintains stem cell pools in the Drosophila midgut, we performed partial ISC depletion. In contrast to the mouse intestine, Drosophila ISCs failed to repopulate the gut after partial depletion. Even when the midgut was challenged to regenerate by infection, ISCs retained normal proportions of asymmetric division and ISC pools did not increase. We discovered, however, that the loss of differentiated midgut enterocytes (ECs) slows when ISC division is suppressed and accelerates when ISC division increases. This plasticity in rates of EC turnover appears to facilitate epithelial homeostasis even after stem cell pools are compromised. Our study identifies unique behaviors of Drosophila midgut cells that maintain epithelial homeostasis.

Hippo vs. Crab: tissue-specific functions of the mammalian Hippo pathway.

The Hippo signaling pathway is a vital suppressor of tumorigenesis that is often inactivated in human cancers. In normal cells, the Hippo pathway is triggered by external forces such as cell crowding, or changes to the extracellular matrix or cell polarity. Once activated, Hippo signaling down-regulates transcription supported by the paralogous cofactors YAP1 and TAZ. The Hippo pathway's functions in normal and cancer biology have been dissected by studies of mutant mice with null or conditional tissue-specific mutations of Hippo signaling elements. In this review, we attempt to systematically summarize results that have been gleaned from detailed invivo characterizations of these mutants. Our goal is to describe the physiological roles of Hippo signaling in several normal organ systems, as well as to emphasize how disruption of the Hippo pathway, and particularly hyperactivation of YAP1/TAZ, can be oncogenic.

Modeling the Effect of Curvature on the Collective Behavior of Cells Growing New Tissue

The growth of several biological tissues is known to be controlled in part by local geometrical features, such as the curvature of the tissue interface. This control leads to changes in tissue shape that in turn can affect the tissue’s evolution. Understanding the cellular basis of this control is highly significant for bioscaffold tissue engineering, the evolution of bone microarchitecture, wound healing, and tumor growth. Although previous models have proposed geometrical relationships between tissue growth and curvature, the role of cell density and cell vigor remains poorly understood. We propose a cell-based mathematical model of tissue growth to investigate the systematic influence of curvature on the collective crowding or spreading of tissue-synthesizing cells induced by changes in local tissue surface area during the motion of the interface. Depending on the strength of diffusive damping, the model exhibits complex growth patterns such as undulating motion, efficient smoothing of irregularities, and the generation of cusps. We compare this model with in vitro experiments of tissue deposition in bioscaffolds of different geometries. By including the depletion of active cells, the model is able to capture both smoothing of initial substrate geometry and tissue deposition slowdown as observed experimentally.

Topological defects in confined populations of spindle-shaped cells

Spindle-shaped cells readily form nematic structures marked by topological defects. When confined, the defect distribution is independent of the domain size, activity and type of cell, lending a stability not found in non-cellular active nematics. Most spindle-shaped cells (including smooth muscles and sarcomas) organize in vivo into well-aligned ‘nematic’ domains1,2,3, creating intrinsic topological defects that may be used to probe the behaviour of these active nematic systems. Active non-cellular nematics have been shown to be dominated by activity, yielding complex chaotic flows4,5. However, the regime in which live spindle-shaped cells operate, and the importance of cell–substrate friction in particular, remains largely unexplored. Using in vitro experiments, we show that these active cellular nematics operate in a regime in which activity is effectively damped by friction, and that the interaction between defects is controlled by the system’s elastic nematic energy. Due to the activity of the cells, these defects behave as self-propelled particles and pairwise annihilate until all displacements freeze as cell crowding increases6,7. When confined in mesoscopic circular domains, the system evolves towards two identical +1/2 disclinations facing each other. The most likely reduced positions of these defects are independent of the size of the disk, the cells’ activity or even the cell type, but are well described by equilibrium liquid crystal theory. These cell-based systems thus operate in a regime more stable than other active nematics, which may be necessary for their biological function.