Cell Migration in 3D microenvironments

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

B.1. In Vitro 3D Vessel Models for Study of Vascular Malformities Reveal Shape Specific Effects On Endothelial Barrier Function. Personnell: Robert Fischer Vascular malformities include tortuous vessels and aneurysms which are hallmarks of myriad disease states, including cancer, cardiovascular diseases, atherosclerosis, strokes, and numerous inherited syndromes. While the mechanisms causing malformations of blood vessels are not well understood, several endothelial pathologies are common to all of them, which include hyperpermeability, activation, inflammation and leukocyte recruitment. In order to understand what effects the physical shape of the vessel has on flow dynamics and endothelial barrier function in the absence of other complicating factors, we use an engineering approach to print blood vessels of defined tortuosity curvature or with aneurysm diameters based on human vessel imaging in a 3D culture model which can be pumped at physiological shear and pulse rates. This system enables us to use high speed imaging to image flow dynamics, as well as monitor local barrier function and endothelial organization. Using this approach, we have found that cells at inner negative curvatures experience altered flow, exhibit decreased coherence, and increased permeability, without the need for external inflammatory signaling. Meanwhile, the turbulent flow and dead zones of aneurysms promotes similar endothelial dysfunction, despite having the opposite curvature sign. Our model system now allows us to probe mechanosensitive pathways to determine how these change in vascular malformities and how they may contribute to overall disease progression, and to model potential treatment strategies using physiological examples of patient vascular disorders. B.2.2. Role of plasma membrane biophysics in establishment of cancer cell polarity during leader bleb-based migration in non-adhesive confinement Personnel: Ankita Jha Collaborators: Jason Haugh, Jared Toettcher Test the hypothesis that front-rear polarity of cancer cells undergoing leader bleb-based migration in non-adhesive confinement is mediated by the segregation of signaling components on the plasma membrane. We found that EGF signaling was required for leader bleb stability under non-adhesive confinement, suggesting the role of plasma membrane growth factor receptors in bleb-based migration. This work shows that during leader bleb-based migration of cancer cells in non-adhesive confinement, transmembrane receptors are depleted from the leading tip of the bleb by strong corralling at the back, likely to maintain persistent bleb polarization. We are currently performing siRNA of CD44 to determine if it is required for leader bled formation and the EGFR gradient, and are utilizing a new photoactivatable EGFR165 to determine if spatial organization of EGF activity is required for polarization of LBBM. Finally, our results predict that cells undergoing leader bleb-based migration may be insensitive to or repulsed by growth factor gradients, and we are gearing up to test this as well. B.4. Project: Upregulation of Lamin B Receptor in Metastatic Melanoma Promotes Enhanced Nuclear Envelope Fragility. Personnel: Michelle Baird Collaborators: Mehdi Pirzoona, Alex Cartegena-Riviera Goal: Determine the molecular pathways leading to nuclear envelope fragility in confined metastatic cancer cells. We found that cell lines derived from advanced prostate or breast cancers or metastatic melanoma all showed enhanced NE fragility compared to cell lines from normal prostate, breast and melanocyte origin. We found that high expression of lamin B receptor (LBR) in metastatic melanoma cells was required for enhanced nuclear envelope fragility in confinement and increased nuclear deformability as measured by AFM. We discovered that LBR over-expression was sufficient to increase NE fragility and deformability in normal melanocytes. Together our results indicate that upregulation of LBR during cancer progression promotes disruption of cholesterol organization in the NE to mediate increased nuclear deformability and fragility in cancer cells subject to confinement, and suggests sterol reductase activity as a possible anti-metastatic drug target. We are currently seeing if LBR expression or its sterol reductase activity are required for NE failure in metastatic melanoma cells in physiological settings including tumor models in vitro and in mice, and whether they promote genetic heterogeneity caused by repeated bouts of confined migration. B.5. Project: The LINC complex mediates Unlocking of Neurectoderm Lineage Gene (Sox1) During Early Differentiation in Mouse Embryonic Stem Cells Personnel: Mehdi Hamouda Collaborators: Kevin Chalut, Kate Miroshnikova Goal: Test the hypothesis that cytoskeletal forces generated in nave pluripotent stem cells during morphogenesis of the peri-implantation blastocyst are transmitted through the LINC complex to epigenetically effect lineage commitment during exit from nave pluripotency. We studied the role of the LINC complex and actomyosin activity in mouse embryonic stem cells (mESCs) in tissue culture during exit from nave pluripotency. These results suggest that morphogenesis-associated apical actomyosin contractile activity may mechanically signal through the LINC complex to instruct appropriate chromatin remodeling at the apical nuclear surface which unlocks Sox1 for timely downstream transcriptional activation. We are currently delving more deeply into the cytoskeletal mechanism, aiming to determine which small GTPase and MRLC kinase directs apical constriction, and whether the barbed-end binding protein emerin plays a role in tethering actin to the NE. B.6 Project: Uncovering The Role Of The NLRP3 Inflammasome In Neutrophil Chemotaxis Personnell: Martina Lerche Collaborators: Denisa D. Wagner Neutrophils are part of the innate immune system and are one of the host’s defense first responders to infections or wounding. Upon encountering infection or stress, the NLRP3 inflammasome, consisting of the sensor protein NLRP3, the ASC adaptor protein and the effector pro-caspase-1, assembles and self-organizes into a protein scaffold at the microtubule organizing center (MTOC). This assembly allows for proximity induced auto processing of pro-caspase-1 to form the catalytically active protease caspase-1. Caspase-1 cleaves the cytokines pro-IL-18 and pro-IL-1ß into their mature and active forms which are released to activate inflammation, and also cleaves gasdermin D, which induces pore formation at the plasma membrane triggering pyroptosis, a pro-inflammatory form of lytic cell death. We have previously characterized the role of the NLRP3 inflammasome in neutrophil polarization and chemotaxis (Van Bruggen et al. 2023). We found that neutrophils from NLRP3-/- mice display an increased cell area, a reduced cell elongation, a defect in centrosome orientation, and lose their capacity to chemotax in response to a leukotriene B4 (LTB4) gradient. Treating wild-type neutrophils with the small molecule inhibitor, MCC950 which blocks the inflammasome assembly, impacted only chemotaxis but not the general movement of neutrophils, indicating inflammasome assembly is required for cell polarization. In mice, lack of NLRP3 led to a reduced infiltration of neutrophils in response to a laser-induced liver burn injury. However, the mechanism by which the NLRP3 inflammasome mediates directed cell migration in neutrophils remains elusive. Here, we set out to determine the mechanism by which the NLRP3 inflammasome regulates chemotaxis of neutrophils by studying the role, and identifying the cleavage substrates of active caspase-1 that mediate this function.