Heterotypic Cell Interactions Research Articles

Exploring the cell interactome: deciphering relative impacts of cell-cell communication in cell co-culture using a novel microfluidic device.

The human body is made up of approximately 40 trillion cells in close contact, with the cellular density of individual tissues varying from 1 million to 1 billion cells per cubic centimetre. Interactions between different cell types (termed heterotypic) are thus common in vivo. Communication between cells can take the form of direct cell-cell contact mediated by plasma membrane proteins or through paracrine signalling mediated through the release, diffusion, and receipt of soluble factors. There is currently no systematic method to investigate the relative contributions of these mechanisms to cell behaviour. In this paper, we detail the conception, development and validation of a microfluidic device that allows cell-cell contact and paracrine signalling in defined areas and over a variety of biologically relevant length scales, referred to as the interactome-device or 'I-device'. Importantly, by intrinsic device design features, cells in different regions in the device are exposed to four different interaction types, including a) no heterotypic cell interaction, b) only paracrine signalling, c) only cell-cell direct contact, or d) both forms of interaction (paracrine and cell-cell direct contact) together. The device design was validated by both mathematical modelling and experiments. Perfused stem cell culture over the medium term and the formation of direct contact between cells in the culture chambers was confirmed. The I-device offers significant flexibility, being able to be applied to any combination of adherent cells to determine the relative contributions of different communication mechanisms to cellular outcomes.

Abstract C056: Characterization of prostate cancer secretomes for therapeutic intervention

Abstract Introduction: Prostate cancer (PC) is a common malignancy among men, often driven by dysregulated androgen receptor (AR) signaling. Earlier stages of PC can be favorably treated with androgen deprivation therapies or AR signaling inhibitors. Unfortunately, metastatic PC (mPC) is currently incurable. Neuroendocrine prostate cancer (NEPC) is a subgroup of mPC that may be derived as result of transdifferentiation of adenocarcinomas, after AR-directed treatment. Previous studies using preclinical models have suggested a potentially beneficial relationship between NEPC and AR-active prostate cancer (ARPC) cells. We hypothesize that secreted factors derived from NEPC cells influence local, and distant ARPC cell types to promote treatment resistance. Methods: Five NEPC in vitro cell models: NCI-H660, MSKCC-EF1, LTL331R, LuCaP-49 and LuCaP-173.1 were maintained in serum free media for 24 hours. The conditioned media (CM) was then collected and admixed with growth medium for ARPC cells. The ARPC growth medium was either supplemented with Fetal Bovine Serum (FBS) or Charcoal Stripped fetal bovine Serum (CSS). GFP-labelled ARPC cells: LnCaP and C42B were seeded in the respective media admixed with the CM. The viability of cells was measured using a Cytation5 microplate reader. We implemented a transcriptomic (RNAseq) and mass-spectrometry based proteomic analysis to identify differentially expressing secretory proteins that could have an impact on the treatment resistance of ARPC cell types. Results: All 5 NEPC-CM showed qualitative growth promoting effects towards ARPC C42B cells grown in CSS supplemented media. Only CM from EF1 and H660 promoted the growth of ARPC C42B in FBS supplemented media. NEPC-CM did not affect growth of LnCaP ARPC cells. Mass-spectrometry for secreted proteins was performed on 3 NEPC cell models. Analysis of the proteins against the PANTHER gene ontology database shows: (i) cell communication, and (ii) cellular response to stimulus, as two important biological processes active in NEPC. Genes involved in the two pathways were further compared against a comprehensive transcriptomic dataset spanning distinct mPC phenotypes and genotypes. Neurotrophic growth factors: Brain-derived growth factor (BDNF), Nerve-growth factor (NGF), VGF and neurotrophin-3 (NT-3) emerged as important targets that were differentially expressed in NEPC versus ARPC cells. Conclusions: NEPC cells have the potential to influence ARPC phenotypes that include enhanced proliferation, AR targeting, and potentially other drug resistance programs. As mPC exhibits substantial intra-individual and intra-tumor heterogeneity, understanding a targeting heterotypic cell interactions could lead to new druggable targets and address treatment resistance resulting from complex inter-cellular interactions. (Funding: PC220408) Citation Format: Tarana Arman, Lisa A. Jones, Ilsa M. Coleman, Philip R. Gafken, Peter S. Nelson. Characterization of prostate cancer secretomes for therapeutic intervention [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C056.

Transcriptional drifts associated with environmental changes in endothelial cells.

Environmental cues, such as physical forces and heterotypic cell interactions play a critical role in cell function, yet their collective contributions to transcriptional changes are unclear. Focusing on human endothelial cells, we performed broad individual sample analysis to identify transcriptional drifts associated with environmental changes that were independent of genetic background. Global gene expression profiling by RNA sequencing and protein expression by liquid chromatography-mass spectrometry directed proteomics distinguished endothelial cells in vivo from genetically matched culture (in vitro) samples. Over 43% of the transcriptome was significantly changed by the in vitro environment. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes. Inclusion of heterotypic interactions by co-culture of endothelial cells with smooth muscle cells normalized approximately 9% of the original in vivo signature. We also identified novel flow dependent genes, as well as genes that necessitate heterotypic cell interactions to mimic the in vivo transcriptome. Our findings highlight specific genes and pathways that rely on contextual information for adequate expression from those that are agnostic of such environmental cues.

Mesenchymal-endothelial nexus in breast cancer spheroids induces vasculogenesis and local invasion in a CAM model

Interplay between non-cancerous cells (immune, fibroblasts, mesenchymal stromal cells (MSC), and endothelial cells (EC)) has been identified as vital in driving tumor progression. As studying such interactions in vivo is challenging, ex vivo systems that can recapitulate in vivo scenarios can aid in unraveling the factors impacting tumorigenesis and metastasis. Using the synthetic tumor microenvironment mimics (STEMs)—a spheroid system composed of breast cancer cells (BCC) with defined human MSC and EC fractions, here we show that EC organization into vascular structures is BC phenotype dependent, and independent of ERα expression in epithelial cancer cells, and involves MSC-mediated Notch1 signaling. In a 3D-bioprinted model system to mimic local invasion, MDA STEMs collectively respond to serum gradient and form invading cell clusters. STEMs grown on chick chorioallantoic membrane undergo local invasion to form CAM tumors that can anastomose with host vasculature and bear the typical hallmarks of human BC and this process requires both EC and MSC. This study provides a framework for developing well-defined in vitro systems, including patient-derived xenografts that recapitulate in vivo events, to investigate heterotypic cell interactions in tumors, to identify factors promoting tumor metastasis-related events, and possibly drug screening in the context of personalized medicine.

Crosstalk between PKR1 and miR-124 is crucial for EMT regulation of tc21+epicardial mesothelial cells during cardiac development and repair process

Abstract Background The epicardial-mesothelial cells (EMCs) are essential regulators of cardiac growth and repairment process. The epicardial-to-mesenchymal transition (EMT) of epicardial fate determination are controlled by both cell autonomous and cardiomyocyte-originated mechanisms. Here, by using an in vitro and in vivo model of epicardial EMT, we investigated the role of miRNAs as regulators of these process and their potential targets. Methods EMT was induced in mice embryonic tcf21+EMCs through an angiogenic cytokine, prokineticin treatments. Cre-dependent tracing of TCF21-tm-iCRE-EMCs was utilized to abrogate prokineticin receptor-1 (PKR1) in mice epicardium. Human tcf21+ cardiac fibroblast (mainly originated from epicardial origin also utilized to study for their repairment signaling. Results Upon EMC-specific abrogation of PKR1 in early stages of cardiac development the TCF21-tm-iCREPKR1−/− mice exhibited 13±4% embryonic lethality due to a disconnection of epicardial cells from compact layer, failed expansion of the sub-epicardial space, and disruption of heterotypic cell interaction between epicardium and myocardium. EMT-RT profiler and enrichment analysis revealed an impaired EMT in TCF21-tm-iCREPKR1−/− hearts. Ingenuity Pathway Analysis on the EMT molecular network (e.g., Snai1, Snai2, B-catenin, N-cadherin, vimentin), specific to cardiac development and defects revealed MiR-124 as a part of this network that was inversely associated and down-stream of PKR1 in cultured Tcf21+ cells. Furthermore, protein expression of SNAIL2 was significantly downregulated in TCF21-tm-iCREPKR1−/− hearts and cultured Tcf21+ cells upon treatment with mir124 mimic. The luciferase reporter assay showed that miR124 directly targeted the 3'-untranslated region of SNAIL2 in EMCs. In a counter experiment, PKR1 gene replacement or mir124 inhibitor was able to rescue the impaired EMT-genes, and an increase in apoptosis and impaired proliferation in epicardium of embryonic TCF21-tm-iCREPKR1−/− hearts. Importantly, miRNA mimic in cultured tcf21 cells maintained epithelial features in EMCs, and increased EMT-associated transcripts and traits, such as apoptosis. In contrast, MiR124 is involved in wound healing process in tcf21+human CFs and EMCs. Conclusion This study shows the importance of a crosstalk between PKR1 and miR-124 with respect to EMT regulation during cardiac development and repair. Controlling fibroblast-committed EMT and pathological fibrotic remodeling is of increasing interest within the field of regenerative tissue engineering and development of interventional strategies after cardiac injury. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Fondation de France

The corticotroph cells from early development to tumorigenesis.

During development, highly specialized differentiated cells, such as pituitary secretory cells, acquire their identity and properties through a series of specification events exerted by transcription factors to implement a unique gene expression program and epigenomic state. The investigation of these developmental processes informs us on the unique features of a cell lineage, both to explain these features and also to outline where these processes may fail and cause disease. This review summarizes present knowledge on the developmental origin of pituitary corticotroph and melanotroph cells and on the underlying molecular mechanisms. At the onset, comparison of gene expression programs active in pituitary progenitors compared to those active in differentiated corticotrophs or melanotrophs indicated dramatic differences in the control of, for example, the cell cycle. Tpit is the transcription factor that determines terminal differentiation of pro-opiomelanocortin (POMC) lineages, both corticotrophs and melanotrophs, and its action involves this switch in cell cycle control in parallel with activation of cell-specific gene expression. There is thus far more to making a corticotroph cell than just activating transcription of the POMC gene. Indeed, Tpit also controls implementation of mechanisms for enhanced protein translation capacity and development of extensive secretory organelles. The corticotroph cell identity also includes mechanisms responsible for homotypic cell-cell interactions between corticotrophs and for privileged heterotypic cell interactions with pituitary cells of other lineages. The review also summarizes current knowledge on how a pioneer transcription factor, Pax7, remodels the epigenome such that the same determination transcription factor, Tpit, will implement the melanotroph program of gene expression. Finally, this canvas of regulatory mechanisms implementing POMC lineage identities constitutes the background to understand alterations that characterize corticotroph adenomas of Cushing's disease patients. The integration of all these data into a unified scheme will likely yield a scheme to globally understand pathogenic mechanisms in Cushing's disease.

Multi‐scale Computational Model of Endothelial Cell‐Pericyte Coupling in Idiopathic Pulmonary Fibrosis

IntroductionMicrovascular stability is highly dependent on endothelial cell‐pericyte coupling. In fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), extracellular matrix stiffening and elevated concentrations of profibrotic factors, such as TGF‐β, can disrupt this cell‐cell communication. Treatment of IPF with the antifibrotic drug Nintedanib, which targets PDGF‐βR, FGF‐R, and VEGF‐R signaling, may counteract these effects to strengthen endothelial cell‐pericyte coupling and promote microvessel stability1,2. We have developed a multi‐scale computational model that simulates intracellular signaling in endothelial cells and pericytes, heterotypic cell‐cell communication, and the dynamic lung microenvironment to study how Nintedanib2 and other drugs affect microvascular network remodeling in IPF. We hypothesize that blocking PDGF‐βR and FGF‐R signaling with Nintedanib is sufficient to rescue TGF‐β‐induced endothelial cell‐pericyte decoupling.Materials and MethodsThe multi‐scale model is comprised of logic‐based ordinary differential equations representing intracellular signaling networks in both endothelial cells and pericytes integrated into an agent‐based model representing the lung environment wherein the simulated cells interact with one another, sensing and dynamically altering their microenvironment (Figure 1). The logic‐based network signaling models were developed using the Netflux3 toolkit in MATLAB. The agent‐based model of the spatiotemporal 2D lung environment was constructed in NetLogo4. The multi‐scale model was created by linking the logic‐based network models with the agent‐based model using the NL4Py5 package in Python.Results and DiscussionEndothelial cell‐pericyte decoupling was signified by decreased N‐cadherin expression in pericytes, increased αSMA and Col1mRNA (indicating possible pericyte‐to‐myofibroblast transition), and increased physical separation and distances between endothelial cells and pericytes. The multi‐scale model predicted that increasing TGF‐β concentrations significantly elevated αSMA and Col1mRNA expression in simulated pericytes. N‐cadherin levels did not change in response to TGF‐β or Nintedanib treatment in the network model of the pericyte alone but were affected by the presence of endothelial cells in the agent‐based model, highlighting the importance of endothelial cell‐pericyte interactions in determining pericyte behaviors.ConclusionsOur multi‐scale model demonstrates the importance of considering endothelial cell‐pericyte communication in predicting cellular responses to pro‐fibrotic environments. By representing intracellular biochemical signaling networks, heterotypic cell interactions, and the dynamic multi‐cell microenvironment, we were able to demonstrate a potential mechanism through which Nintedanib treatment affects endothelial cell‐pericyte coupling in IPF.References1) Hanumegowda, C., et al.; Chest. 2012 2) Wollin, L., et al.; Eur Respir J. 2015 3) Kraeutler, MJ., et al.; BMC Syst Biol. 2010 4) Sklar, E., Artif. Life 2007 5) Gunaratne, C.; 2018. NL4Py.

Identification of Transcriptional Drifts in Endothelial Cells Associated with Environmental Changes

Environmental cues, such as physical forces and heterotypic cell interactions play a critical role in cell function, yet their collective contributions to transcriptional changes are unclear. Focusing on endothelial cells, we performed broad individual sample analysis to identify transcriptional drifts associated with environmental changes that were independent of genetic background. Global gene expression profiling by RNA-seq, ATACseq, and protein expression by LC-MS directed proteomics distinguished endothelial cells in vivo from genetically matched culture (in vitro) samples. Over 43% of the transcriptome was significantly changed by the in vitro environment. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes. In contrast, co-culture of endothelial cells with smooth muscle cells normalized approximately 9% of the original in vivo signature. Our findings highlight specific genes and pathways that rely on contextual information and physical forces.

Correction: A microphysiological early metastatic niche on a chip reveals how heterotypic cell interactions and inhibition of integrin subunit β3 impact breast cancer cell extravasation.

Correction for 'A microphysiological early metastatic niche on a chip reveals how heterotypic cell interactions and inhibition of integrin subunit β3 impact breast cancer cell extravasation' by Martina Crippa et al., Lab Chip, 2021, DOI: .

A microphysiological early metastatic niche on a chip reveals how heterotypic cell interactions and inhibition of integrin subunit β3 impact breast cancer cell extravasation.

During metastatic progression multiple players establish competitive mechanisms, whereby cancer cells (CCs) are exposed to both pro- and anti-metastatic stimuli. The early metastatic niche (EMN) is a transient microenvironment which forms in the circulation during CC dissemination. EMN is characterized by the crosstalk among CCs, platelets, leukocytes and endothelial cells (ECs), increasing CC ability to extravasate and colonize secondary tissues. To better understand this complex crosstalk, we designed a human "EMN-on-a-chip" which involves the presence of blood cells as compared to standard metastases-on-chip models, hence providing a microenvironment more similar to the in vivo situation. We showed that CC transendothelial migration (TEM) was significantly increased in the presence of neutrophils and platelets in the EMN-on-a-chip compared to CC alone. Moreover, exploiting the EMN-on-chip in combination with multi-culture experiments, we showed that platelets increased the expression of epithelial to mesenchymal transition (EMT) markers in CCs and that the addition of a clinically approved antiplatelet drug (eptifibatide, inhibiting integrin β3) impaired platelet aggregation and decreased CC expression of EMT markers. Inhibition of integrin β3 in the co-culture system modulated the activation of the Src-FAK-VE-cadherin signaling axis and partially restored the architecture of inter-endothelial junctions by limiting VE-cadherinY658 phosphorylation and its nuclear localization. These observations correlate with the decreased CC TEM observed in the presence of integrin β3 inhibitor. Our EMN-on-a-chip can be easily implemented for drug repurposing studies and to investigate new candidate molecules counteracting CC extravasation.

Targeting of BCL-2 Family Members during Anticancer Treatment: A Necessary Compromise between Individual Cell and Ecosystemic Responses?

The imbalance between BCL-2 homologues and pro-death counterparts frequently noted in cancer cells endows them with a cell autonomous survival advantage. To eradicate ectopic cells, inhibitors of these homologues (BH3 mimetics) were developed to trigger, during anticancer treatment, full activation of the canonical mitochondrial apoptotic pathway and related caspases. Despite efficiency in some clinical settings, these compounds do not completely fulfill their initial promise. We herein put forth that a growing body of evidence indicates that mitochondrial integrity, controlled by BCL-2 family proteins, and downstream caspases regulate other cell death modes and influence extracellular signaling by committed cells. Moreover, intercellular communications play a key role in spreading therapeutic response across cancer cell populations and in engaging an immune response. We thus advocate that BH3 mimetics administration would be more efficient in the long term if it did not induce apoptosis in all sensitive cells at the same time, but if it could instead allow (or trigger) death signal production by non-terminally committed dying cell populations. The development of such a trade-off strategy requires to unravel the effects of BH3 mimetics not only on each individual cancer cell but also on homotypic and heterotypic cell interactions in dynamic tumor ecosystems.

Vascular elastic fiber heterogeneity in health and disease.

Elastin has historically been described as an amorphous protein that functions to provide recoil to tissues that stretch. However, evidence is growing that elastin's role may not be limited to biomechanics. In this minireview, we will summarize current knowledge regarding vascular elastic fibers, focusing on structural differences along the arterial tree and how those differences may influence the behavior of affiliated cells. Regional heterogeneity, including differences in elastic lamellar number, density and cell developmental origin, plays an important role in vessel health and function. These differences impact cell-cell communication, proliferation and movement. Perturbations of normal cell-matrix interactions are correlated with human diseases including aneurysm, atherosclerosis and hypertension. Although classically described as a structural protein, recent data suggest that differences in elastin deposition along the arterial tree have important effects on heterotypic cell interactions and human disease.

Single-Cell Determination of Cardiac Microtissue Structure and Function Using Light Sheet Microscopy.

Native cardiac tissue is composed of heterogeneous cell populations that work cooperatively for proper tissue function; thus, engineered tissue models have moved toward incorporating multiple cardiac cell types in an effort to recapitulate native multicellular composition and organization. Cardiac tissue models composed of stem cell-derived cardiomyocytes (CMs) require inclusion of non-myocytes to promote stable tissue formation, yet the specific contributions of the supporting non-myocyte population on the parenchymal CMs and cardiac microtissues have to be fully dissected. This gap can be partly attributed to limitations in technologies able to accurately study the individual cellular structure and function that comprise intact three-dimensional (3D) tissues. The ability to interrogate the cell-cell interactions in 3D tissue constructs has been restricted by conventional optical imaging techniques that fail to adequately penetrate multicellular microtissues with sufficient spatial resolution. Light sheet fluorescence microscopy (LSFM) overcomes these constraints to enable single-cell resolution structural and functional imaging of intact cardiac microtissues. Multicellular spatial distribution analysis of heterotypic cardiac cell populations revealed that CMs and cardiac fibroblasts were randomly distributed throughout 3D microtissues. Furthermore, calcium imaging of live cardiac microtissues enabled single-cell detection of CM calcium activity, which showed that functional heterogeneity correlated with spatial location within the tissues. This study demonstrates that LSFM can be utilized to determine single-cell spatial and functional interactions of multiple cell types within intact 3D engineered microtissues, thereby facilitating the determination of structure-function relationships at both tissue-level and single-cell resolution. Impact statement The ability to achieve single-cell resolution by advanced three-dimensional light imaging techniques enables exquisite new investigation of multicellular analyses in native and engineered tissues. In this study, light sheet fluorescence microscopy was used to define structure-function relationships of distinct cell types in engineered cardiac microtissues by determining heterotypic cell distributions and interactions throughout the tissues as well as by assessing regional differences in calcium handing functional properties at the individual cardiomyocyte level.

Notch signaling regulates arterial vasoreactivity through opposing functions of Jagged1 and Dll4 in the vessel wall.

Functional interactions between endothelial cells (ECs) and smooth muscle cells (SMCs) in the arterial wall are necessary for controlling vasoreactivity that underlies vascular resistance and tone. Key signaling pathways converge on the phosphorylation of myosin light chain (p-MLC), the molecular signature of force production in SMCs, through coordinating the relative activities of myosin light chain kinase (MLCK) and myosin phosphatase (MP). Notch signaling in the vessel wall serves critical roles in arterial formation and maturation and has been implicated in arterial vasoregulation. In this report, we hypothesized that Notch signaling through ligands Jagged1 (in SMCs) and delta-like protein-4 (Dll4; in ECs) regulates vasoreactivity via homotypic (SMC-SMC) and heterotypic (EC-SMC) cell interactions. Using ligand induction assays, we demonstrated that Jagged1 selectively induced smooth muscle MLCK gene expression and p-MLC content while inhibiting MP function (i.e., increased Ca2+ sensitization) in a Rho kinase II-dependent manner. Likewise, selective deficiency of smooth muscle Jagged1 in mice resulted in MLCK and p-MLC loss, reduced Ca2+ sensitization, and impaired arterial force generation measured by myography. In contrast, smooth muscle Notch signaling triggered by Dll4 increased expression of MP-targeting subunit 1 (MYPT1; the MP regulatory subunit), whereas arteries from endothelial Dll4-deficient mice featured reduced MYPT1 levels, enhanced force production, and impaired relaxation independent of endothelium-derived nitric oxide signaling. Taken together, this study identifies novel opposing vasoregulatory functions for ligand-specific Notch signaling in the vessel wall, underscoring instructional signaling between ECs and SMCs and suggesting that Notch signals might behave as a "rheostat" in arterial tone control. NEW & NOTEWORTHY The present study unveils novel roles for ligand-specific Notch signaling in arterial function. Smooth muscle Jagged1 and endothelial cell delta-like protein-4 ligands exhibit selective regulation of myosin light chain kinase and myosin phosphatase-targeting subunit 1/myosin phosphatase, respectively, providing a mechanistic link through which Notch signals modulate contractile activities in vascular smooth muscle. These findings may inform vascular derangements observed in human syndromes of Notch signaling deficiency while offering fundamental molecular insights into arterial physiological function.

Cardiomyocyte-Endothelial Cell Interactions in Cardiac Remodeling and Regeneration.

The heart is a complex organ consisting of various cell types, each of which plays an important role in both physiological and pathophysiological conditions. The cells communicate with each other through direct cell-cell interactions and paracrine signaling, and both homotypic and heterotypic cell interactions contribute to the organized structure and proper function of the heart. Cardiomyocytes (CMs) and endothelial cells (ECs) are two of the most abundant cardiac cell types and they also play central roles in both cardiac remodeling and regeneration. The postnatal cell cycle withdrawal of CMs, which takes place within days or weeks after birth, represents the major barrier for regeneration in adult mammalian hearts, as adult CMs exhibit a very low proliferative capacity. Recent evidence highlights the importance of ECs not only as the most abundant cell type in the heart but also as key players in post-infarction remodeling and regeneration. In this MiniReview, we focus on blood vascular ECs and CMs and their roles and interactions in cardiac physiology and pathologies, with a special emphasis on cardiac regeneration. We summarize the known mediators of the bidirectional CM-EC interactions and discuss the related recent advances in the development of therapies aiming to promote heart repair and regeneration targeting these two cell types.

An Optimized 3D Coculture Assay for Preclinical Testing of Pro- and Antiangiogenic Drugs

Angiogenesis is a promising target for anticancer therapies, but also for treating other diseases with pathologic vessel development. Targeting the vascular endothelial growth factor (VEGF) pathway did not proof as effective as expected due to emerging intrinsic resistance mechanisms, as well as stromal contributions leading to drug insensitivity. Therefore, alternative strategies affecting the interaction of endothelial cells (ECs) with other stromal cells seem to be more promising. Human preclinical in vitro angiogenesis models successfully recapitulating these interactions are rare, and two-dimensional (2D) cell cultures cannot mimic tissue architecture in vivo. Consequently, models combining three-dimensionality with heterotypic cell interaction seem to be better suited. Here, we report on an improved human fibroblast–EC coculture assay mimicking sprouting angiogenesis from EC-covered microbeads resembling existing endothelial structures. Culture conditions were optimized to assess pro- and antiangiogenic compounds. Important characteristics of angiogenesis, that is, the number of sprouts and branch points, sprout length protrusion, and overall vessel structure areas, were quantified. Notably, the endothelial sprouts display lumen formation and basal membrane establishment. In this model, angiogenesis can be inhibited by genetic interference of pro-angiogenic factors expressed in the fibroblasts. Moreover, bona fide antiangiogenic drugs decreased, whereas pro-angiogenic factors increased vessel formation in 24-well and 96-well settings, demonstrating the applicability for screening approaches.

Development and characterization of a microfluidic model of the tumour microenvironment.

The physical microenvironment of tumours is characterized by heterotypic cell interactions and physiological gradients of nutrients, waste products and oxygen. This tumour microenvironment has a major impact on the biology of cancer cells and their response to chemotherapeutic agents. Despite this, most in vitro cancer research still relies primarily on cells grown in 2D and in isolation in nutrient- and oxygen-rich conditions. Here, a microfluidic device is presented that is easy to use and enables modelling and study of the tumour microenvironment in real-time. The versatility of this microfluidic platform allows for different aspects of the microenvironment to be monitored and dissected. This is exemplified here by real-time profiling of oxygen and glucose concentrations inside the device as well as effects on cell proliferation and growth, ROS generation and apoptosis. Heterotypic cell interactions were also studied. The device provides a live ‘window’ into the microenvironment and could be used to study cancer cells for which it is difficult to generate tumour spheroids. Another major application of the device is the study of effects of the microenvironment on cellular drug responses. Some data is presented for this indicating the device’s potential to enable more physiological in vitro drug screening.

Cellular interactions regulate stem cell differentiation in tri-culture

ABSTRACTCurrently, the mechanism governing the regeneration of the soft tissue-to-bone interface, such as the transition between the anterior cruciate ligament (ACL) and bone, is not known. Focusing on the ACL-to-bone insertion, this study tests the novel hypothesis that interactions between cells from the ligament (fibroblasts) and bone (osteoblasts) initiate interface regeneration. Specifically, these heterotypic cell interactions direct the fibrochondrogenic differentiation of interface-relevant cell populations, defined here as ligament fibroblasts and bone marrow stromal cells (BMSC). The objective of this study is to examine the effects of heterotypic cellular interactions on BMSC or fibroblast growth and biosynthesis, as well as expression of fibrocartilage-relevant markers in tri-culture. The effects of cell–cell physical contact and paracrine interactions between fibroblasts and osteoblasts were also determined. It was found that, in tri-culture with fibroblasts and osteoblasts, BMSC exhibited greater fibrochondrogenic potential than ligament fibroblasts. The growth of BMSC decreased while proteoglycan production and TGF-β3 expression increased. Moreover, tri-culture regulated BMSC response via paracrine factors, and interestingly, fibroblast–osteoblast contact further promoted proteoglycan and TGF-β1 synthesis as well as induced SOX9 expression in BMSC. Collectively, the findings of this study suggest that fibroblast–osteoblast interactions play an important role in regulating the stem cell niche for fibrocartilage regeneration, and the mechanisms of these interactions are directed by paracrine factors and augmented with direct cell–cell contact.

Perivascular Macrophages Limit Permeability.

Perivascular cells, including pericytes, macrophages, smooth muscle cells, and other specialized cell types, like podocytes, participate in various aspects of vascular function. However, aside from the well-established roles of smooth muscle cells and pericytes, the contributions of other vascular-associated cells are poorly understood. Our goal was to ascertain the function of perivascular macrophages in adult tissues under nonpathological conditions. We combined confocal microscopy, in vivo cell depletion, and in vitro assays to investigate the contribution of perivascular macrophages to vascular function. We found that resident perivascular macrophages are associated with capillaries at a frequency similar to that of pericytes. Macrophage depletion using either clodronate liposomes or antibodies unexpectedly resulted in hyperpermeability. This effect could be rescued when M2-like macrophages, but not M1-like macrophages or dendritic cells, were reconstituted in vivo, suggesting subtype-specific roles for macrophages in the regulation of vascular permeability. Furthermore, we found that permeability-promoting agents elicit motility and eventual dissociation of macrophages from the vasculature. Finally, in vitro assays showed that M2-like macrophages attenuate the phosphorylation of VE-cadherin upon exposure to permeability-promoting agents. This study points to a direct contribution of macrophages to vessel barrier integrity and provides evidence that heterotypic cell interactions with the endothelium, in addition to those of pericytes, control vascular permeability.

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics' cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.