Dynamics of multicellular swirling on micropatterned substrates.

Chirality plays a crucial role in biology, as it is highly conserved and fundamentally important in the developmental process. To better understand the relationship between the chirality of individual cells and that of tissues and organisms, we develop a generalized mechanics model of chiral polarized particles to investigate the swirling dynamics of cell populations on substrates. Our analysis reveals that cells with the same chirality can form distinct chiral patterns on ring-shaped or rectangular substrates. Interestingly, our studies indicate that an excessively strong or weak individual cellular chirality hinders the formation of such chiral patterns. Our studies also indicate that there exists the influence distance of substrate boundaries in chiral patterns. Smaller influence distances are observed when cell-cell interactions are weaker. Conversely, when cell-cell interactions are too strong, multiple cells tend to be stacked together, preventing the formation of chiral patterns on substrates in our analysis. Additionally, we demonstrate that the interaction between cells and substrate boundaries effectively controls the chiral distribution of cellular orientations on ring-shaped substrates. This research highlights the significance of coordinating boundary features, individual cellular chirality, and cell-cell interactions in governing the chiral movement of cell populations and provides valuable mechanics insights into comprehending the intricate connection between the chirality of single cells and that of tissues and organisms.

Using random forests to uncover the predictive power of distance-varying cell interactions in tumor microenvironments.

Tumor microenvironments (TMEs) contain vast amounts of information on patient's cancer through their cellular composition and the spatial distribution of tumor cells and immune cell populations. Exploring variations in TMEs between patient groups, as well as determining the extent to which this information can predict outcomes such as patient survival or treatment success with emerging immunotherapies, is of great interest. Moreover, in the face of a large number of cell interactions to consider, we often wish to identify specific interactions that are useful in making such predictions. We present an approach to achieve these goals based on summarizing spatial relationships in the TME using spatial K functions, and then applying functional data analysis and random forest models to both predict outcomes of interest and identify important spatial relationships. This approach is shown to be effective in simulation experiments at both identifying important spatial interactions while also controlling the false discovery rate. We further used the proposed approach to interrogate two real data sets of Multiplexed Ion Beam Images of TMEs in triple negative breast cancer and lung cancer patients. The methods proposed are publicly available in a companion R package funkycells.

The angiotensin II receptors type 1 and 2 modulate astrocytes and their crosstalk with microglia and neurons in an in vitro model of ischemic stroke.

BACKGROUND: Astrocytes are the most abundant cell type of the central nervous system and are fundamentally involved in homeostasis, neuroprotection, and synaptic plasticity. This regulatory function of astrocytes on their neighboring cells in the healthy brain is subject of current research. In the ischemic brain we assume disease specific differences in astrocytic acting. The renin-angiotensin-aldosterone system regulates arterial blood pressure through endothelial cells and perivascular musculature. Moreover, astrocytes express angiotensin II type 1 and 2 receptors. However, their role in astrocytic function has not yet been fully elucidated. We hypothesized that the angiotensin II receptors impact astrocyte function as revealed in an in vitro system mimicking cerebral ischemia. Astrocytes derived from neonatal wistar rats were exposed to telmisartan (angiotensin II type 1 receptor-blocker) or PD123319 (angiotensin II type 2 receptor-blocker) under normal conditions (control) or deprivation from oxygen and glucose. Conditioned medium (CM) of astrocytes was harvested to elucidate astrocyte-mediated indirect effects on microglia and cortical neurons. RESULT: The blockade of angiotensin II type 1 receptor by telmisartan increased the survival of astrocytes during ischemic conditions in vitro without affecting their proliferation rate or disturbing their expression of S100A10, a marker of activation. The inhibition of the angiotensin II type 2 receptor pathway by PD123319 resulted in both increased expression of S100A10 and proliferation rate. The CM of telmisartan-treated astrocytes reduced the expression of pro-inflammatory mediators with simultaneous increase of anti-inflammatory markers in microglia. Increased neuronal activity was observed after treatment of neurons with CM of telmisartan- as well as PD123319-stimulated astrocytes. CONCLUSION: Data show that angiotensin II receptors have functional relevance for astrocytes that differs in healthy and ischemic conditions and effects surrounding microglia and neuronal activity via secretory signals. Above that, this work emphasizes the strong interference of the different cells in the CNS and that targeting astrocytes might serve as a therapeutic strategy to influence the acting of glia-neuronal network in de- and regenerative context.

Unusual cell-cell cooperative mechanical activity elucidates the process of tissue regeneration.

We have studied wound contraction in three model wounds in animals: excised skin (guinea pig), transected peripheral nerve (rat) and the excised conjunctiva (rabbit). Wound contraction is driven by myofibroblasts bound together by adherens junctions (AJ) that confer cooperative activity to myofibroblasts during wound contraction and synthesis of scar. Grafting with the dermis regeneration template (DRT) cancels cell cooperativity by abolishing AJ connections in myofibroblasts, while also cancelling wound contraction, preventing synthesis of scar and inducing regeneration of excised tissues. The observed definitive prevention of scar synthesis suggests the exploration of DRT scaffolds to regenerate tissues in several other organs and to prevent fibrosis in humans.

interFLOW: maximum flow framework for the identification of factors mediating the signaling convergence of multiple receptors.

Cell-cell crosstalk involves simultaneous interactions of multiple receptors and ligands, followed by downstream signaling cascades working through receptors converging at dominant transcription factors, which then integrate and propagate multiple signals into a cellular response. Single-cell RNAseq of multiple cell subsets isolated from a defined microenvironment provides us with a unique opportunity to learn about such interactions reflected in their gene expression levels. We developed the interFLOW framework to map the potential ligand-receptor interactions between different cell subsets based on a maximum flow computation in a network of protein-protein interactions (PPIs). The maximum flow approach further allows characterization of the intracellular downstream signal transduction from differentially expressed receptors towards dominant transcription factors, therefore, enabling the association between a set of receptors and their downstream activated pathways. Importantly, we were able to identify key transcription factors toward which the convergence of multiple receptor signaling pathways occurs. These identified factors have a unique role in the integration and propagation of signaling following specific cell-cell interactions.

The science of exosomes: Understanding their formation, capture, and role in cellular communication.

Extracellular vesicles (EVs) serve as a crucial method for transferring information among cells, which is vital in multicellular organisms. Among these vesicles, exosomes are notable for their small size, ranging from 20 to 150 nm, and their role in cell-to-cell communication. They carry lipids, proteins, and nucleic acids between cells. The creation of exosomes begins with the inward budding of the cell membrane, which then encapsulates various macromolecules as cargo. Once filled, exosomes are released into the extracellular space and taken up by target cells via endocytosis and similar processes. The composition of exosomal cargo varies, encompassing diverse macromolecules with specific functions. Because of their significant roles, exosomes have been isolated from various cell types, including cancer cells, endothelial cells, macrophages, and mesenchymal cells, with the aim of harnessing them for therapeutic applications. Exosomes influence cellular metabolism, and regulate lipid, glucose, and glutamine pathways. Their role in pathogenesis is determined by their cargo, which can manipulate processes such as apoptosis, proliferation, inflammation, migration, and other molecular pathways in recipient cells. Non-coding RNA transcripts, a common type of cargo, play a pivotal role in regulating disease progression. Exosomes are implicated in numerous biological and pathological processes, including inflammation, cancer, cardiovascular diseases, diabetes, wound healing, and ischemic-reperfusion injury. As a result, they hold significant potential in the treatment of both cancerous and non-cancerous conditions.

Characterizing dysregulations via cell-cell communications in Alzheimer's brains using single-cell transcriptomes.

BACKGROUND: Alzheimer's disease (AD) is a devastating neurodegenerative disorder affecting 44 million people worldwide, leading to cognitive decline, memory loss, and significant impairment in daily functioning. The recent single-cell sequencing technology has revolutionized genetic and genomic resolution by enabling scientists to explore the diversity of gene expression patterns at the finest resolution. Most existing studies have solely focused on molecular perturbations within each cell, but cells live in microenvironments rather than in isolated entities. Here, we leveraged the large-scale and publicly available single-nucleus RNA sequencing in the human prefrontal cortex to investigate cell-to-cell communication in healthy brains and their perturbations in AD. We uniformly processed the snRNA-seq with strict QCs and labeled canonical cell types consistent with the definitions from the BRAIN Initiative Cell Census Network. From ligand and receptor gene expression, we built a high-confidence cell-to-cell communication network to investigate signaling differences between AD and healthy brains. RESULTS: Specifically, we first performed broad communication pattern analyses to highlight that biologically related cell types in normal brains rely on largely overlapping signaling networks and that the AD brain exhibits the irregular inter-mixing of cell types and signaling pathways. Secondly, we performed a more focused cell-type-centric analysis and found that excitatory neurons in AD have significantly increased their communications to inhibitory neurons, while inhibitory neurons and other non-neuronal cells globally decreased theirs to all cells. Then, we delved deeper with a signaling-centric view, showing that canonical signaling pathways CSF, TGFß, and CX3C are significantly dysregulated in their signaling to the cell type microglia/PVM and from endothelial to neuronal cells for the WNT pathway. Finally, after extracting 23 known AD risk genes, our intracellular communication analysis revealed a strong connection of extracellular ligand genes APP, APOE, and PSEN1 to intracellular AD risk genes TREM2, ABCA1, and APP in the communication from astrocytes and microglia to neurons. CONCLUSIONS: In summary, with the novel advances in single-cell sequencing technologies, we show that cellular signaling is regulated in a cell-type-specific manner and that improper regulation of extracellular signaling genes is linked to intracellular risk genes, giving the mechanistic intra- and inter-cellular picture of AD.

Extracellular vesicles in endometriosis: role and potential.

Endometriosis is a chronic inflammatory gynecological disease, which profoundly jeopardizes women's quality of life and places a significant medical burden on society. The pathogenesis of endometriosis remains unclear, posing major clinical challenges in diagnosis and treatment. There is an urgent demand for the development of innovative non-invasive diagnostic techniques and the identification of therapeutic targets. Extracellular vesicles, recognized for transporting a diverse array of signaling molecules, have garnered extensive attention as a novel mode of intercellular communication. A burgeoning body of research indicates that extracellular vesicles play a pivotal role in the pathogenesis of endometriosis, which may provide possibility and prospect for both diagnosis and treatment. In light of this context, this article focuses on the involvement of extracellular vesicles in the pathogenesis of endometriosis, which deliver information among endometrial stromal cells, macrophages, mesenchymal stem cells, and other cells, and explores their potential applications in the diagnosis and treatment, conducing to the emergence of new strategies for clinical diagnosis and treatment.

Connexin channels and hemichannels are modulated differently by charge reversal at residues forming the intracellular pocket.

BACKGROUND: Members of the ß-subfamily of connexins contain an intracellular pocket surrounded by amino acid residues from the four transmembrane helices. The presence of this pocket has not previously been investigated in members of the α-, γ-, δ-, and ε-subfamilies. We studied connexin50 (Cx50) as a representative of the α-subfamily, because its structure has been determined and mutations of Cx50 are among the most common genetic causes of congenital cataracts. METHODS: To investigate the presence and function of the intracellular pocket in Cx50 we used molecular dynamics simulation, site-directed mutagenesis, gap junction tracer intercellular transfer, and hemichannel activity detected by electrophysiology and by permeation of charged molecules. RESULTS: Employing molecular dynamics, we determined the presence of the intracellular pocket in Cx50 hemichannels and identified the amino acids participating in its formation. We utilized site-directed mutagenesis to alter a salt-bridge interaction that supports the intracellular pocket and occurs between two residues highly conserved in the connexin family, R33 and E162. Substitution of opposite charges at either position decreased formation of gap junctional plaques and cell-cell communication and modestly reduced hemichannel currents. Simultaneous charge reversal at these positions produced plaque-forming non-functional gap junction channels with highly active hemichannels. CONCLUSIONS: These results show that interactions within the intracellular pocket influence both gap junction channel and hemichannel functions. Disruption of these interactions may be responsible for diseases associated with mutations at these positions.

Stem Cell-Derived Extracellular Vesicles: Promising Therapeutic Opportunities for Diabetic Wound Healing.

Wound healing is a sophisticated and orderly process of cellular interactions in which the body restores tissue architecture and functionality following injury. Healing of chronic diabetic wounds is difficult due to impaired blood circulation, a reduced immune response, and disrupted cellular repair mechanisms, which are often associated with diabetes. Stem cell-derived extracellular vesicles (SC-EVs) hold the regenerative potential, encapsulating a diverse cargo of proteins, RNAs, and cytokines, presenting a safe, bioactivity, and less ethical issues than other treatments. SC-EVs orchestrate multiple regenerative processes by modulating cellular communication, increasing angiogenesis, and promoting the recruitment and differentiation of progenitor cells, thereby potentiating the reparative milieu for diabetic wound healing. Therefore, this review investigated the effects and mechanisms of EVs from various stem cells in diabetic wound healing, as well as their limitations and challenges. Continued exploration of SC-EVs has the potential to revolutionize diabetic wound care.

Signalling switches maintain intercellular communication in the vascular endothelium.

BACKGROUND AND PURPOSE: The single layer of cells lining all blood vessels, the endothelium, is a sophisticated signal co-ordination centre that controls a wide range of vascular functions including the regulation of blood pressure and blood flow. To co-ordinate activities, communication among cells is required for tissue level responses to emerge. While a significant form of communication occurs by the propagation of signals between cells, the mechanism of propagation in the intact endothelium is unresolved. EXPERIMENTAL APPROACH: Precision signal generation and targeted cellular manipulation was used in conjunction with high spatiotemporal mesoscale Ca2+ imaging in the endothelium of intact blood vessels. KEY RESULTS: Multiple mechanisms maintain communication so that Ca2+ wave propagation occurs irrespective of the status of connectivity among cells. Between adjoining cells, regenerative IP3-induced IP3 production transmits Ca2+ signals and explains the propagated vasodilation that underlies the increased blood flow accompanying tissue activity. The inositide is itself sufficient to evoke regenerative phospholipase C-dependent Ca2+ waves across coupled cells. None of gap junctions, Ca2+ diffusion or the release of extracellular messengers is required to support this type of intercellular Ca2+ signalling. In contrast, when discontinuities exist between cells, ATP released as a diffusible extracellular messenger transmits Ca2+ signals across the discontinuity and drives propagated vasodilation. CONCLUSION AND IMPLICATIONS: These results show that signalling switches underlie endothelial cell-to-cell signal transmission and reveal how communication is maintained in the face of endothelial damage. The findings provide a new framework for understanding wave propagation and cell signalling in the endothelium.

Protocol for unsupervised inference of cell-cell communication using matrix decomposition.

Exploring cell-cell communication is pivotal for understanding biological processes in multicellular life forms. Here, we present a protocol that details the use of matrix decomposition to infer cell-cell communication (MDIC3) for unsupervised cell-cell communication inference. We describe steps for using the MDIC3 Python scripts to deduce cell-cell communication and identify key ligand-receptor pairs between a specific cell type pair from a single-cell gene expression dataset. This protocol has potential application in cell-cell communication inference on any species. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

A resource-based mechanistic framework for castration-resistant prostate cancer (CRPC).

Cancer therapy often leads to the selective elimination of drug-sensitive cells from the tumour. This can favour the growth of cells resistant to the therapeutic agent, ultimately causing a tumour relapse. Castration-resistant prostate cancer (CRPC) is a well-characterised instance of this phenomenon. In CRPC, after systemic androgen deprivation therapy (ADT), a subset of drug-resistant cancer cells autonomously produce testosterone, thus enabling tumour regrowth. A previous theoretical study has shown that such a tumour relapse can be delayed by inhibiting the growth of drug-resistant cells using biotic competition from drug-sensitive cells. In this context, the centrality of resource dynamics to intra-tumour competition in the CRPC system indicates clear scope for the construction of theoretical models that can explicitly incorporate the underlying mechanisms of tumour ecology. In the current study, we use a modified logistic framework to model cell-cell interactions in terms of the production and consumption of resources. Our results show that steady state composition of CRPC can be understood as a composite function of the availability and utilisation efficiency of two resources-oxygen and testosterone. In particular, we show that the effect of changing resource availability or use efficiency is conditioned by their general abundance regimes. Testosterone typically functions in trace amounts and thus affects steady state behaviour of the CRPC system differently from oxygen, which is usually available at higher levels. Our data thus indicate that explicit consideration of resource dynamics can produce novel and useful mechanistic understanding of CRPC. Furthermore, such a modelling approach also incorporates variables into the system's description that can be directly measured in a clinical context. This is therefore a promising avenue of research in cancer ecology that could lead to therapeutic approaches that are more clearly rooted in the biology of CRPC.

The potential roles of exosomes in pathological cardiomyocyte hypertrophy mechanisms and therapy: A review.

Pathological cardiac hypertrophy, characterized by the enlargement of cardiac muscle cells, leads to serious cardiac conditions and stands as a major global health issue. Exosomes, comprising small lipid bilayer vesicles, are produced by various cell types and found in numerous bodily fluids. They play a pivotal role in intercellular communication by transferring bioactive cargos to recipient cells or activating signaling pathways in target cells. Exosomes from cardiomyocytes, endothelial cells, fibroblasts, and stem cells are key in regulating processes like cardiac hypertrophy, cardiomyocyte survival, apoptosis, fibrosis, and angiogenesis within the context of cardiovascular diseases. This review delves into exosomes' roles in pathological cardiac hypertrophy, first elucidating their impact on cell communication and signaling pathways. It then advances to discuss how exosomes affect key hypertrophic processes, including metabolism, fibrosis, oxidative stress, and angiogenesis. The review culminates by evaluating the potential of exosomes as biomarkers and their significance in targeted therapeutic strategies, thus emphasizing their critical role in the pathophysiology and management of cardiac hypertrophy.

The Landscape of Exosomes Biogenesis to Clinical Applications.

Exosomes are extracellular vesicles that originate from various cells and mediate intercellular communication, altering the behavior or fate of recipient cells. They carry diverse macromolecules, such as lipids, proteins, carbohydrates, and nucleic acids. Environmental stressors can change the exosomal contents of many cells, making them useful for diagnosing many chronic disorders, especially neurodegenerative, cardiovascular, cancerous, and diabetic diseases. Moreover, exosomes can be engineered as therapeutic agents to modulate disease processes. State-of-art techniques are employed to separate exosomes including ultracentrifugation, size-exclusion chromatography and immunoaffinity. However, modern technologies such as aqueous two-phase system as well as microfluidics are gaining attention in the recent years. The article highlighted the composition, biogenesis, and implications of exosomes, as well as the standard and novel methods for isolating them and applying them as biomarkers and therapeutic cargo carriers.

Exosomal mediators in sepsis and inflammatory organ injury: unraveling the role of exosomes in intercellular crosstalk and organ dysfunction.

Sepsis, a severe systemic inflammatory response to infection, remains a leading cause of morbidity and mortality worldwide. Exosomes, as mediators of intercellular communication, play a pivotal role in the pathogenesis of sepsis through modulating immune responses, metabolic reprogramming, coagulopathy, and organ dysfunction. This review highlights the emerging significance of exosomes in these processes. Initially, it provides an in-depth insight into exosome biogenesis and characterization, laying the groundwork for understanding their diverse and intricate functions. Subsequently, it explores the regulatory roles of exosomes in various immune cells such as neutrophils, macrophages, dendritic cells, T cells, and B cells. This analysis elucidates how exosomes are pivotal in modulating immune responses, thus contributing to the complexity of sepsis pathophysiology. Additionally, this review delves into the role of exosomes in the regulation of metabolism and subsequent organ dysfunction in sepsis. It also establishes a connection between exosomes and the coagulation cascade, which affects endothelial integrity and promotes thrombogenesis in sepsis. Moreover, the review discusses the dual role of exosomes in the progression and resolution of sepsis, exploring their complex involvement in inflammation and healing processes. Furthermore, it underscores their potential as biomarkers and therapeutic targets. Understanding these mechanisms presents new opportunities for novel interventions to mitigate the severe outcomes of sepsis, emphasizing the therapeutic promise of exosome research in critical care settings.

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk. hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function. hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Combining LIANA and Tensor-cell2cell to decipher cell-cell communication across multiple samples.

In recent years, data-driven inference of cell-cell communication has helped reveal coordinated biological processes across cell types. Here, we integrate two tools, LIANA and Tensor-cell2cell, which, when combined, can deploy multiple existing methods and resources to enable the robust and flexible identification of cell-cell communication programs across multiple samples. In this work, we show how the integration of our tools facilitates the choice of method to infer cell-cell communication and subsequently perform an unsupervised deconvolution to obtain and summarize biological insights. We explain how to perform the analysis step by step in both Python and R and provide online tutorials with detailed instructions available at https://ccc-protocols.readthedocs.io/. This workflow typically takes ∼1.5 h to complete from installation to downstream visualizations on a graphics processing unit-enabled computer for a dataset of ∼63,000 cells, 10 cell types, and 12 samples.

Cx31.1 can selectively intermix with co-expressed connexins to facilitate its assembly into gap junctions.

Connexins are channel-forming proteins that function to facilitate gap junctional intercellular communication. Here, we use dual cell voltage clamp and dye transfer studies to corroborate past findings showing that Cx31.1 (encoded by GJB5) is defective in gap junction channel formation, illustrating that Cx31.1 alone does not form functional gap junction channels in connexin-deficient mammalian cells. Rather Cx31.1 transiently localizes to the secretory pathway with a subpopulation reaching the cell surface, which is rarely seen in puncta reminiscent of gap junctions. Intracellular retained Cx31.1 was subject to degradation as Cx31.1 accumulated in the presence of proteasomal inhibition, had a faster turnover when Cx43 was present and ultimately reached lysosomes. Although intracellularly retained Cx31.1 was found to interact with Cx43, this interaction did not rescue its delivery to the cell surface. Conversely, the co-expression of Cx31 dramatically rescued the assembly of Cx31.1 into gap junctions where gap junction-mediated dye transfer was enhanced. Collectively, our results indicate that the localization and functional status of Cx31.1 is altered through selective interplay with co-expressed connexins, perhaps suggesting Cx31.1 is a key regulator of intercellular signaling in keratinocytes.