Eicosanoid Transcellular Biosynthesis: From Cell-Cell Interactions to in Vivo Tissue Responses

2006 ◽  
Vol 58 (3) ◽  
pp. 375-388 ◽  
Author(s):  
Giancarlo Folco ◽  
Robert C. Murphy
Keyword(s):  
Cell Interactions ◽  
Tissue Responses ◽  
Cell Cell

scholarly journals Targeting Rac-Gtpases Reverses Stroma-Induced Resistance to Notch and mTOR-Inhibition in Acute T-Cell Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1326-1326
Author(s):  
Adrian Schwarzer ◽  
Martin May ◽  
Harald Genth ◽  
Zhixiong Li ◽  
Christopher Baum ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Notch Signaling ◽  
Induced Resistance ◽  
Stromal Cells ◽  
Cell Interactions ◽  
Mtor Inhibition ◽  
Rac Gtpases ◽  
Notch Inhibition ◽  
Cell Cell

Abstract Molecular hallmarks of T-ALL are the aberrant activation of NOTCH signaling and high activity of the PI3K-AKT-mTOR pathway. Upregulated mTOR and NOTCH have been linked to the resistance of T-ALL to chemotherapy as well as to high frequencies of leukemia-initiating cells. Hence, the mTOR and the NOTCH pathways are promising therapeutic targets in T-ALL. However, clinical success of the mTOR inhibitor Rapamycin in acute leukemia has been disappointing. Similar results have been observed in mouse models of T-ALL treated with Notch inhibitors. To investigate the impact of mTOR and Notch inhibition in a genetically complex T-ALL, we developed an aggressive murine T-ALL model, driven by tyrosine kinase signaling, loss of Pten, Cux1-haploinsufficiency and constitutive Notch signaling. In vitro, T-ALL blasts were highly sensitive to inhibition of AKT, mTOR and Notch signaling. We transplanted the leukemias into secondary recipients and initiated treatment with Rapamycin after the onset of leukemia. Rapamycin significantly prolonged survival of the animals (placebo: 27 days, Rapamycin 49 days, p<0.001). Eventually, all Rapamycin treated animals succumbed to the T-ALL that extensively infiltrated the bone marrow and solid organs despite continuous drug administration. When Rapamycin-resistant blasts were explanted and cultured in petri dishes they again became susceptible to Rapamycin, demonstrating a context-dependent resistance rather than outgrowth of intrinsically resistant clones. Gene set enrichment analysis revealed that Rapamycin-resistant T-ALL in vivo upregulated genetic networks associated with cell-cell interactions. Stromal cell support from OP9-cells as well as from mesenchymal stem cells recapitulated the in vivo effect and induced resistance to mTOR and Notch-inhibition in T-ALL blasts. Coating the tissue culture wells with Collagen, Fibronectin, Retronectin or Matrigel, did not elicit resistance. By using trans-well assays we show that the stroma-induced resistance was dependent on direct cell-cell interactions. Immunoblots and PhosFlow probing the mTORC1/C2 and Notch pathway demonstrated an identical drug effect on their intracellular targets in resistant T-ALL blasts cultured on stroma cells and susceptible cells in suspension. Since the number of molecules potentially involved in cell-to-cell contacts is vast, we focused on central nodes that organize this process in order to find a potentially druggable target that is critically involved in stroma-induced resistance. Transcriptome profiling pointed towards upregulation of Rac-associated pathways. We determined the activation of Rac1 by PAK-pull down assays in T-ALL blasts grown in suspension or on stromal cells. We observed an increase (FC=1.96 ± 0.58, p=0.04) in activated Rac1 in the T-ALL blasts in contact with a stromal layer. To determine whether Rac activation plays a role in stroma-induced resistance, we devised a strategy to abrogate Rac signaling in T-ALL blasts, but not in the stromal cells, since inhibition of Rac in stromal cells by the Rac-inhibitor NSC23766 led to the their detachment. Furthermore, Rac1,2 and 3 can be functionally redundant, making knock down experiments using shRNAs challenging. The Clostridium difficile serotype F strain 1470 produces toxin B isoform (TcdBF), that selectively glucosylates and inactivates Rac(1,2,3). We pretreated T-ALL blasts with TcdBF and observed a dose-dependent functional inhibition of Rac GTPases monitored by dephosphorylation of the Rac effector kinase pS144/141-PAK-1/2. T-ALL blasts were then incubated for 5 hours with increasing toxin doses, washed 3 times and incubated in toxin-free medium. Eighteen hours after the end of the exposure to the toxins, Rac was still inhibited. Strikingly, in the TcdBF-pretreated T-ALL, the stroma-induced resistance effect was abrogated and clusters of apoptotic cells were clearly visible (>2 fold reduction of the input, p=0.002). In contrast, the carrier-treated T-ALL exhibited resistance to the inhibitors on stroma (>10 fold expansion of the input, p<0.0003). Altogether, we identify the Rac-GTPases as a nexus of stroma-induced drug resistance and show that inhibition of Rac and mTOR is synthetically lethal to T-ALL blasts T-ALL blasts that are in contact with stromal cells, paving the way to augment the effectiveness of small molecule inhibitors in acute leukemia. Disclosures No relevant conflicts of interest to declare.

Anergic T cells exert antigen-independent inhibition of cell-cell interactions via chemokine metabolism

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2173-2179 ◽  
Author(s):  
Martha J. James ◽  
Lavina Belaramani ◽  
Kanella Prodromidou ◽  
Arpita Datta ◽  
Sussan Nourshargh ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
T Cell Activation ◽  
Cell Interactions ◽  
Cell Surface Expression ◽  
Anergic T Cells ◽  
Parenchymal Cells ◽  
Cell Cell

Abstract Due to their ability to inhibit antigen-induced T-cell activation in vitro and in vivo, anergic T cells can be considered part of the spectrum of immunoregulatory T lymphocytes. Here we report that both murine and human anergic T cells can impair the ability of parenchymal cells (including endothelial and epithelial cells) to establish cell-cell interactions necessary to sustain leukocyte migration in vitro and tissue infiltration in vivo. The inhibition is reversible and cell-contact dependent but does not require cognate recognition of the parenchymal cells to occur. Instrumental to this effect is the increased cell surface expression and enzymatic activity of molecules such as CD26 (dipeptidyl-peptidase IV), which may act by metabolizing chemoattractants bound to the endothelial/epithelial cell surface. These results describe a previously unknown antigen-independent anti-inflammatory activity by locally generated anergic T cells and define a novel mechanism for the long-known immunoregulatory properties of these cells.

Reduction of TGF-beta activity abrogates growth promoting tumor cell-cell interactions in vivo

1991 ◽  
Vol 148 (3) ◽  
pp. 380-390 ◽  
Author(s):  
D. Theodorescu ◽  
M. Caltabiano ◽  
R. Greig ◽  
D. Rieman ◽  
R. S. Kerbel
Keyword(s):  
Tumor Cell ◽  
Cell Interactions ◽  
Beta Activity ◽  
Tgf Beta ◽  
Growth Promoting ◽  
Cell Cell

scholarly journals Formation of organotypic testicular organoids in microwell culture†

2019 ◽  
Vol 100 (6) ◽  
pp. 1648-1660 ◽  
Author(s):  
Sadman Sakib ◽  
Aya Uchida ◽  
Paula Valenzuela-Leon ◽  
Yang Yu ◽  
Hanna Valli-Pulaski ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Germ Cell ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Cell Interactions ◽  
Dependent Manner ◽  
Tissue Architecture ◽  
Cell Cell

Abstract Three-dimensional (3D) organoids can serve as an in vitro platform to study cell–cell interactions, tissue development, and toxicology. Development of organoids with tissue architecture similar to testis in vivo has remained a challenge. Here, we present a microwell aggregation approach to establish multicellular 3D testicular organoids from pig, mouse, macaque, and human. The organoids consist of germ cells, Sertoli cells, Leydig cells, and peritubular myoid cells forming a distinct seminiferous epithelium and interstitial compartment separated by a basement membrane. Sertoli cells in the organoids express tight junction proteins claudin 11 and occludin. Germ cells in organoids showed an attenuated response to retinoic acid compared to germ cells in 2D culture indicating that the tissue architecture of the organoid modulates response to retinoic acid similar to in vivo. Germ cells maintaining physiological cell–cell interactions in organoids also had lower levels of autophagy indicating lower levels of cellular stress. When organoids were treated with mono(2-ethylhexyl) phthalate (MEHP), levels of germ cell autophagy increased in a dose-dependent manner, indicating the utility of the organoids for toxicity screening. Ablation of primary cilia on testicular somatic cells inhibited the formation of organoids demonstrating an application to screen for factors affecting testicular morphogenesis. Organoids can be generated from cryopreserved testis cells and preserved by vitrification. Taken together, the testicular organoid system recapitulates the 3D organization of the mammalian testis and provides an in vitro platform for studying germ cell function, testicular development, and drug toxicity in a cellular context representative of the testis in vivo.

scholarly journals Vascular Geometry and Flow Profile Mediate Pathological Cell-Cell Interactions in Sickle Cell Disease As Measured with "Do-It-Yourself" "Endothelial-Ized" Microfluidics

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 454-454
Author(s):  
Robert Mannino ◽  
David R Myers ◽  
Byungwook Ahn ◽  
Hope Gole ◽  
Yichen Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Cell Adhesion ◽  
Cell Interactions ◽  
Cell Disease ◽  
In Vivo Models ◽  
Do It Yourself ◽  
Cell Cell

Abstract Background and Significance: Cell-cell interactions between blood cells and endothelial cells play an important role in sickle cell disease (SCD) pathophysiology. While in vivo transgenic animal models and in vitro systems have both contributed to our understanding of these pathologic cell-cell interactions in SCD, isolating the causes and effects of cellular interactions is exceedingly difficult in the former and recapitulating the complex vascular geometries found in vivo is not readily available with current systems in the latter. The vascular system comprises diverse geometries that range from normal (e.g. curves and bifurcations) to pathologic (e.g. aneurysms and stenoses) and as blood flows from one vascular geometry to another, the local shear stress profile acutely changes. Interestingly, changes in shear stress are known to alter endothelial pro-inflammatory signaling pathways and expression of cell adhesion molecules, especially vascular cell adhesion molecule-1(VCAM-1) (Tzima, Nature, 2005), which is implicated in SCD vasculopathy. Here we present a rapid and inexpensive method using only off-the-shelf materials to create “do-it-yourself” (DIY) microfluidic devices that incorporate endothelial cells and clinically relevant vascular geometries; this system effectively and bridges current in vitro and in vivo models to study SCD. Using this technique, we developed a vascularized bifurcation, and observed that shear stress changes can be extremely localized, affecting only several 10s of cells, and are associated with changes in VCAM1 expression. We used this in vitro vascularized bifurcation to test the hypothesis that SS RBC-endothelial cell adhesion occurs primarily at bifurcations, which are difficult to visualize in vivo (Nagel, Arterioscler Thromb Vasc Biol, 1999). We demonstrate that SCD RBCs do primarily aggregate at bifurcations, specifically in locations where the shear stress has decreased and VCAM-1 is upregulated. Methods: In order to bridge in vitro data with the complex vascular geometric environments found in vivo, we developed a “DIY” endothelialized microfluidic model (Figure 1A). A strand of 500um diameter polymethylmethacrylate (PMMA) optical fiber is laid flat on top of a layer of polydimethylsiloxane (PDMS) and covered with a second, thin layer of PDMS. After curing, the optical fiber is pulled out, exposing a hollow, circular, channel that can be used as a microchannel to seed endothelial cells. A wide variety of endothelial cells can be successfully seeded in these devices, such as human umbilical vein endothelial cells, human aortic endothelial cells, and human microvascular endothelial cells. Slight alterations to this fabrication method result in the creation of multiple vascular geometries, such as curved or bifurcated channels with or without aneurysms or stenoses. Results: Curved channels & bifurcations (Figure 1B-C) are seeded with endothelial cells (Figure 1E-F). Computational fluidic dynamics calculations show that the shear varies by 2.5 fold within the bifurcation. As shear affects endothelial expression, we tested if the extremely localized shear changes created in this system were sufficient to alter local endothelial expression of VCAM-1 Indeed, in our system, VCAM1 expression significantly correlated with shear variation (Figure1G), and was highest near the bifurcation point. Noting this localized variation in adhesion molecule expression, we tested whether the bifurcations are implicated in SCD RBC adhesion to the endothelium. With our vascularized bifurcation model and custom image analysis software that quantifies RBC aggregation, we observed that SCD RBC adhesion predominantly occurred at the point of bifurcation where the shear is lowest and VCAM1 expression is greatest, and minimal endothelial adhesion occurred with healthy control RBCs (Figure 2). This phenomenon persisted with tumor necrosis factor-stimulation of the endothelium. Conclusion: This DIY system represents an easily accessible technique that allows any researcher to bridge the gap between in vitro and in vivo models of pathological cell-cell interactions in SCD. We demonstrate that recapitulating the complex vascular geometries in vivo is vital to understanding blood cell-endothelial interactions and this system will not only be useful for studying SCD, but a myriad of hematologic and vascular diseases as well. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Mechanisms of dorsal-ventral patterning in noggin-induced neural tissue

Development ◽  
1997 ◽  
Vol 124 (12) ◽  
pp. 2477-2488 ◽  
Author(s):  
A.K. Knecht ◽  
R.M. Harland
Keyword(s):  
Neural Tissue ◽  
Bmp Signaling ◽  
Cell Interactions ◽  
Dependent Manner ◽  
Low Doses ◽  
Morphogenetic Protein ◽  
High Doses ◽  
Dose Dependent ◽  
Cell Cell

We have investigated mechanisms of dorsal-ventral patterning of neural tissue, using Xenopus ectoderm neuralized by noggin protein. This tissue appears to be patterned dorsoventrally; cp1-1, a gene expressed in the dorsal brain, and etr-1, a gene largely excluded from the dorsal brain, are expressed in separate territories in noggin-treated explants (Knecht, A. K., Good, P. J., Dawid, I. B. and Harland, R. M. (1995) Development 121, 1927–1936). Here we show further evidence that this pattern represents a partial dorsal-ventral organization. Additionally, we test two mechanisms that could account for this pattern: a dose-dependent response to a gradient of noggin protein within the explant, and regulative cell-cell interactions. We show that noggin exhibits concentration-dependent effects, inducing cp1-1 at low doses but repressing it at high doses. Since noggin acts by antagonizing Bone Morphogenetic Protein (BMP) signaling, this result suggests that BMPs also may act in a dose-dependent manner in vivo. However, in the absence of a noggin gradient, regulative cell-cell interactions can also pattern the tissue. Such regulation is facilitated by increased motility of noggin-treated cells. Finally, the response of cells to both of these patterning mechanisms is ultimately controlled by a third process, the changing competence of the responding tissue.

Cell Patterning: Interaction of Cardiac Myocytes and Fibroblasts in Three-Dimensional Culture

2008 ◽  
Vol 14 (2) ◽  
pp. 117-125 ◽  
Author(s):  
Troy A. Baudino ◽  
Alex McFadden ◽  
Charity Fix ◽  
Joshua Hastings ◽  
Robert Price ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Three Dimensional ◽  
Normal Growth ◽  
Cell Types ◽  
Cell Interactions ◽  
Cellular Interactions ◽  
Cell Layers ◽  
Cell Cell

Patterning of cells is critical to the formation and function of the normal organ, and it appears to be dependent upon internal and external signals. Additionally, the formation of most tissues requires the interaction of several cell types. Indeed, both extracellular matrix (ECM) components and cellular components are necessary for three-dimensional (3-D) tissue formationin vitro. Using 3-D cultures we demonstrate that ECM arranged in an aligned fashion is necessary for the rod-shaped phenotype of the myocyte, and once this pattern is established, the myocytes were responsible for the alignment of any subsequent cell layers. This is analogous to thein vivopattern that is observed, where there appears to be minimal ECM signaling, rather formation of multicellular patterns is dependent upon cell–cell interactions. Our 3-D culture of myocytes and fibroblasts is significant in that it modelsin vivoorganization of cardiac tissue and can be used to investigate interactions between fibroblasts and myocytes. Furthermore, we used rotational cultures to examine cellular interactions. Using these systems, we demonstrate that specific connexins and cadherins are critical for cell–cell interactions. The data presented here document the feasibility of using these systems to investigate cellular interactions during normal growth and injury.

scholarly journals PDGF, TGF-β, and Heterotypic Cell–Cell Interactions Mediate Endothelial Cell–induced Recruitment of 10T1/2 Cells and Their Differentiation to a Smooth Muscle Fate

1998 ◽  
Vol 141 (3) ◽  
pp. 805-814 ◽  
Author(s):  
Karen K. Hirschi ◽  
Stephanie A. Rohovsky ◽  
Patricia A. D'Amore
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Shape Change ◽  
Muscle Cells ◽  
Cell Interactions ◽  
Mural Cell ◽  
Cells Cultured ◽  
Cell Cell

We aimed to determine if and how endothelial cells (EC) recruit precursors of smooth muscle cells and pericytes and induce their differentiation during vessel formation. Multipotent embryonic 10T1/2 cells were used as presumptive mural cell precursors. In an under-agarose coculture, EC induced migration of 10T1/2 cells via platelet-derived growth factor BB. 10T1/2 cells in coculture with EC changed from polygonal to spindle-shaped, reminiscent of smooth muscle cells in culture. Immunohistochemical and Western blot analyses were used to examine the expression of smooth muscle (SM)-specific markers in 10T1/2 cells cultured in the absence and presence of EC. SM-myosin, SM22α, and calponin proteins were undetectable in 10T1/2 cells cultured alone; however, expression of all three SM-specific proteins was significantly induced in 10T1/2 cells cocultured with EC. Treatment of 10T1/2 cells with TGF-β induced phenotypic changes and changes in SM markers similar to those seen in the cocultures. Neutralization of TGF-β in the cocultures blocked expression of the SM markers and the shape change. To assess the ability of 10T1/2 cells to contribute to the developing vessel wall in vivo, prelabeled 10T1/2 cells were grown in a collagen matrix and implanted subcutaneously into mice. The fluorescently marked cells became incorporated into the medial layer of developing vessels where they expressed SM markers. These in vitro and in vivo observations shed light on the cell–cell interactions that occur during vessel development, as well as in pathologies in which developmental processes are recapitulated.

scholarly journals Studying 3D cell cultures in a microfluidic droplet array under multiple time-resolved conditions

2018 ◽  
Author(s):  
Raphaël F.-X. Tomasi ◽  
Sébastien Sart ◽  
Tiphaine Champetier ◽  
Charles N. Baroud
Keyword(s):  
Single Cell ◽  
Cell Cultures ◽  
Single Cell Analysis ◽  
Cell Interactions ◽  
Multiple Time ◽  
Time Resolved ◽  
Cell Functions ◽  
Wide Range ◽  
Cell Cell

The relevance of traditional cell cultures to cellular behavior in vivo is limited, since the two-dimensional (2D) format does not appropriately reproduce the microenvironment that regulates cell functions. In this context, spheroids are an appealing 3D cell culture format to complement standard techniques, by combining a high level of biological relevance with simple production protocols. However the methods for spheroid manipulation are still labor intensive, which severely limits the complexity of operations that can be performed on statistically relevant numbers of individual spheroids. Here we show how to apply hundreds of different conditions on spheroids in a single microfluidic chip, where each spheroid is produced and immobilized in an anchored droplet. By using asymmetric anchor shapes, a second drop can be merged with the spheroid-containing drop at a later time. This time-delayed merging uniquely enables two classes of applications that we demonstrate: (1) the initiation of cell-cell interactions on demand, either for building micro-tissues within the device or for observing antagonistic cell-cell interactions with applications in immuno-therapy or host-pathogen interactions, (2) a detailed dose-response curve obtained by exposing an array of hepatocyte-like spheroids to droplets containing a wide range of acetaminophen concentrations. The integrated microfluidic format allows time-resolved measurements of the response of hundreds of spheroids with a single-cell resolution. The data shows an internally regulated evolution of each spheroid, in addition to a heterogeneity of the responses to the drug that the single-cell analysis correlates with the initial presence and location of dead cells within each spheroid.

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