scholarly journals Phagocytic glioblastoma-associated microglia and macrophages populate invading pseudopalisades

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Elena Saavedra-López ◽  
Meritxell Roig-Martínez ◽  
George P Cribaro ◽  
Paola V Casanova ◽  
José M Gallego ◽  
...  
Keyword(s):  
Cell Invasion ◽  
Immune Cells ◽  
Three Dimensional ◽  
Glioma Cells ◽  
Confocal Imaging ◽  
Cellular Motility ◽  
Microfluidic Platforms ◽  
Necrotic Focus ◽  
Hypoxic Environment

Abstract Hypoxic pseudopalisades are a pathological hallmark of human glioblastoma, which is linked to tumour malignancy and aggressiveness. Yet, their function and role in the tumour development have scarcely been explored. It is thought that pseudopalisades are formed by malignant cells escaping from the hypoxic environment, although evidence of the immune component of pseudopalisades has been elusive. In the present work, we analyse the immunological constituent of hypoxic pseudopalisades using high-resolution three-dimensional confocal imaging in tissue blocks from excised tumours of glioblastoma patients and mimic the hypoxic gradient in microfluidic platforms in vitro to understand the cellular motility. We visualize that glioblastoma-associated microglia and macrophages abundantly populate pseudopalisades, displaying an elongated kinetic morphology across the pseudopalisades, and are oriented towards the necrotic focus. In vitro experiments demonstrate that under hypoxic gradient, microglia show a particular motile behaviour characterized by the increase of cellular persistence in contrast with glioma cells. Importantly, we show that glioblastoma-associated microglia and macrophages utilize fibres of glioma cells as a haptotactic cue to navigate along the anisotropic structure of the pseudopalisades and display a high phagocytic activity at the necrotic border of the pseudopalisades. In this study, we demonstrate that glioblastoma-associated microglia and macrophages are the main immune cells of pseudopalisades in glioblastoma, travelling to necrotic areas to clear the resulting components of the prothrombotic milieu, suggesting that the scavenging features of glioblastoma-associated microglia and macrophages at the pseudopalisades serve as an essential counterpart for glioma cell invasion.

scholarly journals Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Marloes I. Hofstee ◽  
Martijn Riool ◽  
Igors Terjajevs ◽  
Keith Thompson ◽  
Martin J. Stoddart ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Cells ◽  
Early Stage ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Maximum Size ◽  
Human Neutrophils ◽  
Content Type

ABSTRACT Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

scholarly journals TWIST1 Heterodimerization with E12 Requires Coordinated Protein Phosphorylation to Regulate Periostin Expression

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1392
Author(s):  
Svetlana A. Mikheeva ◽  
Nathan D. Camp ◽  
Lei Huang ◽  
Antrix Jain ◽  
Sung Yun Jung ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Invasion ◽  
Glioma Cells ◽  
Therapeutic Strategies ◽  
Bhlh Transcription Factor ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Dismal Prognosis ◽  
Adjacent Brain

Diffuse invasion into adjacent brain matter by glioblastoma (GBM) is largely responsible for their dismal prognosis. Previously, we showed that the TWIST1 (TW) bHLH transcription factor and its regulated gene periostin (POSTN) promote invasive phenotypes of GBM cells. Since TW functional effects are regulated by phosphorylation and dimerization, we investigated how phosphorylation of serine 68 in TW regulates TW dimerization, POSTN expression, and invasion in glioma cells. Compared with wild-type TW, the hypophosphorylation mutant, TW(S68A), impaired TW heterodimerization with the E12 bHLH transcription factor and cell invasion in vitro but had no effect on TW homodimerization. Overexpression of TW:E12 forced dimerization constructs (FDCs) increased glioma cell invasion and upregulated pro-invasive proteins, including POSTN, in concert with cytoskeletal reorganization. By contrast, TW:TW homodimer FDCs inhibited POSTN expression and cell invasion in vitro. Further, phosphorylation of analogous PXSP phosphorylation sites in TW:E12 FDCs (TW S68 and E12 S139) coordinately regulated POSTN and PDGFRa mRNA expression. These results suggested that TW regulates pro-invasive phenotypes in part through coordinated phosphorylation events in TW and E12 that promote heterodimer formation and regulate downstream targets. This new mechanistic understanding provides potential therapeutic strategies to inhibit TW-POSTN signaling in GBM and other cancers.

scholarly journals Optical Cellular Micromotion: A New Paradigm to Measure Tumour Cells Invasion in 3D Tumour Environments

2021 ◽  
Author(s):  
Zhaobin Guo ◽  
Chih-Tsung Yang ◽  
Chia-Chi Chien ◽  
Luke Selth ◽  
Pierre Bagnaninchi ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Invasion ◽  
Tumour Cell ◽  
Single Cells ◽  
Three Dimensional ◽  
Tumour Cells ◽  
Digital Holographic Microscopy ◽  
New Paradigm ◽  
Cell Invasiveness

Measuring tumour cell invasiveness through three-dimensional (3D) tissues, particularly at the single cell level, can provide important mechanistic understanding and assist in identifying therapeutic targets of tumour invasion. However, current experimental approaches, including standard in vitro invasion assays, have limited physiological relevance and offer insufficient insight about the vast heterogeneity in tumour cell migration through tissues. To address these issues, here we report on the concept of optical cellular micromotion, where digital holographic microscopy (DHM) is used to map the optical thickness fluctuations at sub-micron scale within single cells. These fluctuations are driven by the dynamic movement of subcellular structures including the cytoskeleton and inherently associated with the biological processes involved in cell invasion within tissues. We experimentally demonstrate that the optical cellular micromotion correlates with tumour cells motility and invasiveness both at the population and single cell levels. In addition, the optical cellular micromotion significantly reduced upon treatment with migrastatic drugs that inhibit tumour cell invasion. These results demonstrate that micromotion measurements can rapidly and non-invasively determine the invasive behaviour of single tumour cells within tissues, yielding a new and powerful tool to assess the efficacy of approaches targeting tumour cell invasiveness.

Geldanamycin inhibits migration of glioma cells in vitro: A potential role for hypoxia-inducible factor (HIF-1?) in glioma cell invasion

2003 ◽  
Vol 196 (2) ◽  
pp. 394-402 ◽  
Author(s):  
David Zagzag ◽  
Motohiro Nomura ◽  
David R. Friedlander ◽  
CY Blanco ◽  
Jean-Pierre Gagner ◽  
...  
Keyword(s):  
Cell Invasion ◽  
Glioma Cell ◽  
Potential Role ◽  
Hypoxia Inducible Factor ◽  
Glioma Cells

scholarly journals Injection of T3SS effectors not resulting in invasion is the main targeting mechanism ofShigellatoward human lymphocytes

2017 ◽  
Vol 114 (37) ◽  
pp. 9954-9959 ◽  
Author(s):  
Laurie Pinaud ◽  
Fatoumata Samassa ◽  
Ziv Porat ◽  
Mariana L. Ferrari ◽  
Ilia Belotserkovsky ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Cell Invasion ◽  
Immune Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Host Cells ◽  
Mucosal Barrier ◽  
Functional Consequences ◽  
B And T Lymphocytes

The enteroinvasive bacteriumShigellais a facultative intracellular bacterium known, in vitro, to invade a large diversity of cells through the delivery of virulence effectors into the cell cytoplasm via a type III secretion system (T3SS). Here, we provide evidence that the injection of T3SS effectors does not necessarily result in cell invasion. Indeed, we demonstrate through optimization of a T3SS injection reporter that effector injection without subsequent cell invasion, termed the injection-only mechanism, is the main strategy used byShigellato target human immune cells. We show that in vitro-activated human peripheral blood B, CD4+T, and CD8+T lymphocytes as well as switched memory B cells are mostly targeted by the injection-only mechanism. B and T lymphocytes residing in the human colonic lamina propria, encountered byShigellaupon its crossing of the mucosal barrier, are also mainly targeted by injection-only. These findings reveal that cells refractory to invasion can still be injected, thus extending the panel of host cells manipulated to the benefit of the pathogen. Future analysis of the functional consequences of the injection-only mechanism toward immune cells will contribute to the understanding of the priming of adaptive immunity, which is known to be altered during the course of naturalShigellainfection.

scholarly journals Mesenchymal-like glioma cells are enriched in the gelatin methacrylate hydrogels

2021 ◽  
Author(s):  
Nameeta Shah ◽  
Pavan M. Hallur ◽  
Raksha A. Ganesh ◽  
Pranali Sonpatki ◽  
Divya Naik ◽  
...  
Keyword(s):  
Therapeutic Response ◽  
Immune Cell ◽  
Three Dimensional ◽  
Glioma Cells ◽  
Cellular Phenotype ◽  
3D Scaffold ◽  
Promising Alternative ◽  
Cells Cultured

AbstractGlioblastoma is the most lethal primary malignant brain tumor in adults. Simplified two-dimensional (2D) cell culture and neurospheres in vitro models fail to recapitulate the complexity of the tumor microenvironment, limiting its ability to predict therapeutic response. Three-dimensional (3D) scaffold-based models have emerged as a promising alternative for addressing these concerns. One such 3D system is gelatin methacrylate (GelMA) hydrogels, which can be used for modeling the glioblastoma microenvironment. We characterized the phenotype of patient-derived glioma cells cultured in GelMA hydrogels (3D-GMH) for their tumorigenic properties using invasion and chemoresponse assays. In addition, we used integrated single-cell and spatial transcriptome analysis to compare cells cultured in 3D-GMH to cells in vivo. Finally, we assessed tumor-immune cell interactions with a macrophage infiltration assay and a cytokine array. We show that cells cultured in 3D-GMH develop a mesenchymal-like cellular phenotype found in perivascular and hypoxic regions present in the core of the tumor, and recruit macrophages by secreting cytokines in contrast to the cells grown as neurospheres that match the phenotype of cells of the infiltrative edge of the tumor.

Abstract 109: IL-1ß-Induced Ceramide Synthesis Accentuates NETosis and Inflammation in Abdominal Aortic Aneurysms

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Akshaya K Meher ◽  
Nicolas Pope ◽  
Gang Su ◽  
John P Davis ◽  
Vlad Serbulea ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Aortic Aneurysms ◽  
Confocal Imaging ◽  
Knock Out ◽  
Liquid Chromatography Tandem Mass ◽  
Ceramide Synthesis ◽  
Aortic Media ◽  
Immunohistochemical Techniques ◽  
Long Chain

Objective: We have previously shown that circulating neutrophils are required for experimental aortic aneurysm (AA) formation via a non-MMP2/9-mediated mechanism. In other experiments we documented that IL-1β signaling is required for AA formation. Therefore, we hypothesized that increased levels of IL-1β in AA triggers inflammatory ceramide synthesis and thus extracellular trap (NET) formation (NETosis) by neutrophils, thereby augmenting inflammation. Methods: Immunohistochemical techniques were used to identify neutrophils and NETs. Ceramides from human AA and control aortas from transplant donors were quantified using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Neutrophils isolated from human blood were treated with IL-1β to determine if IL-1β induces ceramide synthesis and NETosis in vitro. Experimental AAs were induced by elastase perfusion in C57BL/6 wild-type (WT) and IL-1β knock-out (KO) mice and cellular content of AA was analyzed using flow cytometry. Groups were compared using two-tailed unpaired t-test. Results: Immunohistochemical staining showed that neutrophils were scattered or grouped near lymphoid-like structures in the adventitia of human AA. Three-dimensional confocal imaging showed NETs in the adventitia of human AA. Importantly, the levels of long chain ceramides C16 and C24:1 were higher (2.6 and 1.8-fold, respectively), whereas, the levels of short chain ceramides C4, C6 and C8 were lower (4.3, 3.9 and 3.5-fold, respectively) in human AA compared to controls. In isolated neutrophils, IL-1β treatment induced synthesis of long chain ceramides and discharge of NETs. Neutrophils and NETs were also detected in aortic media at day 7 after induction of AA in WT mice. On day 7 following elastase perfusion, despite the increase in number of circulating immune cells, AA size was smaller (91.08±11.7% vs 53.8±5.4%, p=0.03), and, infiltration of neutrophils (12179±2697 vs 3046±628, p=0.01) and B cells was lower in the aorta of IL-1β KO mice compared to WT mice. Conclusions: These data document for the first time the presence of NETs in human AAs. Furthermore, the data suggests that neutrophils augment inflammation in AA by increasing pro-inflammatory long chain ceramide synthesis and NETosis in response to IL-1β.

scholarly journals Gelatin methacrylate hydrogels culture model for glioblastoma cells enriches for mesenchymal-like state and models interactions with immune cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nameeta Shah ◽  
Pavan M. Hallur ◽  
Raksha A. Ganesh ◽  
Pranali Sonpatki ◽  
Divya Naik ◽  
...  
Keyword(s):  
Immune Cell ◽  
Three Dimensional ◽  
Glioma Cells ◽  
Glioblastoma Cells ◽  
Treatment Resistant ◽  
3D Scaffold ◽  
Promising Alternative ◽  
Cells Cultured

AbstractGlioblastoma is the most lethal primary malignant brain tumor in adults. Simplified two-dimensional (2D) cell culture and neurospheres in vitro models fail to recapitulate the complexity of the tumor microenvironment, limiting its ability to predict therapeutic response. Three-dimensional (3D) scaffold-based models have emerged as a promising alternative for addressing these concerns. One such 3D system is gelatin methacrylate (GelMA) hydrogels, and we aimed to understand the suitability of using this system to mimic treatment-resistant glioblastoma cells that reside in specific niches. We characterized the phenotype of patient-derived glioma cells cultured in GelMA hydrogels (3D-GMH) for their tumorigenic properties using invasion and chemoresponse assays. In addition, we used integrated single-cell and spatial transcriptome analysis to compare cells cultured in 3D-GMH to neoplastic cells in vivo. Finally, we assessed tumor-immune cell interactions with a macrophage infiltration assay and a cytokine array. We show that the 3D-GMH system enriches treatment-resistant mesenchymal cells that are not represented in neurosphere cultures. Cells cultured in 3D-GMH resemble a mesenchymal-like cellular phenotype found in perivascular and hypoxic regions and recruit macrophages by secreting cytokines, a hallmark of the mesenchymal phenotype. Our 3D-GMH model effectively mimics the phenotype of glioma cells that are found in the perivascular and hypoxic niches of the glioblastoma core in situ, in contrast to the neurosphere cultures that enrich cells of the infiltrative edge of the tumor. This contrast highlights the need for due diligence in selecting an appropriate model when designing a study‘s objectives.

Quantitative Evaluation of Melanoma Cell Invasion in Three-Dimensional Confrontation Cultures In Vitro Using Automated Image Analysis

1990 ◽  
Vol 94 (1) ◽  
pp. 114-119 ◽  
Author(s):  
Josef Smolle ◽  
Christine Helige ◽  
Hans-Peter Soyer ◽  
Stefan Hoedl ◽  
Helmut Popper ◽  
...  
Keyword(s):  
Image Analysis ◽  
Quantitative Evaluation ◽  
Melanoma Cell ◽  
Cell Invasion ◽  
Three Dimensional ◽  
Automated Image Analysis
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