A novel workflow for three-dimensional analysis of tumour cell migration

Ketchen, Sophie; Rohwedder, Arndt; Knipp, Sabine; Esteves, Filomena; Struve, Nina; Peckham, Michelle; Ladbury, John E.; Curd, Alistair; Short, Susan; Brüning-Richardson, Anke

doi:10.1098/rsfs.2019.0070

Cited by 5 publications

(14 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To assess whether GRB2 influences the clustering of FGFR2, we used an unbiased method for identifying the presence of discrete clusters of FGFR2 on the plasma membrane. For this we developed a plugin tool for ImageJ that detected the plasma membrane and quantified the fluorescently labelled protein-containing clusters localised within (modified from a cell surface morphology tool 27 (Cluster 19 plugin. Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Composition of receptor tyrosine kinase-mediated lipid micro-domains controlled by adaptor protein interaction

Rohwedder

Knipp

Roberts

et al. 2021

Sci Rep

Self Cite

View full text Add to dashboard Cite

Receptor tyrosine kinases (RTKs) are highly regulated, single pass transmembrane proteins, fundamental to cellular function and survival. Aberrancies in regulation lead to corruption of signal transduction and a range of pathological outcomes. Although control mechanisms associated with the receptors and their ligands are well understood, little is known with respect to the impact of lipid/lipid and lipid/protein interactions in the proximal plasma membrane environment. Given that the transmembrane regions of RTKs change in response to extracellular ligand binding, the lipid interactions have important consequences in influencing signal transduction. Fibroblast growth factor receptor 2 (FGFR2) is a highly regulated RTK, including under basal conditions. Binding of the adaptor protein, growth factor receptor-bound protein 2 (GRB2) to FGFR2 prevents full activation and recruitment of downstream signalling effector proteins in the absence of extracellular stimulation. Here we demonstrate that the FGFR2-GRB2 complex is sustained in a defined lipid environment. Dissociation of GRB2 from this complex due to ligand binding, or reduced GRB2 expression, facilitates the dispersion of FGFR2 into detergent-resistant membrane (DRM) micro-domains. This modification of the plasma membrane proximal to FGFR2 provides a further regulatory checkpoint which controls receptor degradation, recycling and recruitment of intracellular signalling proteins.

show abstract

Section: Resultsmentioning

confidence: 99%

Composition of receptor tyrosine kinase-mediated lipid micro-domains controlled by adaptor protein interaction

Rohwedder

Knipp

Roberts

et al. 2021

Sci Rep

Self Cite

View full text Add to dashboard Cite

show abstract

“…The contributions come from members of a Medical Research Council (MRC) funded network, 3DBioNet, which aims to establish multidisciplinary teams of scientists from industry and academia who together possess the diverse skills needed to develop 3D micro-tissues. As explained below, the six featured articles span mathematical and computational modelling [3], bioreactor design [4], organoid development [5] and image acquisition and analysis [6].Several papers in this theme issue are devoted to perfusion bioreactors, recognizing that flow bioreactors provide a more realistic environment within which to study cells. For example, in [7], attention focuses on the use of perfusion bioreactors to assess the chemical safety and efficacy of drugs.…”

mentioning

confidence: 99%

“…In [6], attention focuses on a new 3D technique for imaging cell migration within 3D multicellular spheroids, the method being designed to resolve the limitations of existing methods, which typically use 2D images to infer migration patterns in three dimensions. The demand for more accurate techniques is driven largely by an increase in the development of antimigratory drugs designed to inhibit the spread of highly invasive cancers such as gliomas.…”

mentioning

confidence: 99%

“…The demand for more accurate techniques is driven largely by an increase in the development of antimigratory drugs designed to inhibit the spread of highly invasive cancers such as gliomas. The technique proposed by Ketchen et al [6] is based on instant structured illuminated microscopy (iSIM): image capture at high spatio-temporal resolution enables rapid capture of high-resolution volumes (Zstacks). They have used their technique to acquire 3D images of individual cells migrating within labelled multicellular spheroids embedded in collagen.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Three-dimensional biological cultures and organoids

Byrne

2020

Interface Focus.

View full text Add to dashboard Cite

One contribution of 7 to a theme issue '3D biological cultures and organoids'.While two-dimensional (2D) cell models and animal models have played central roles in advancing knowledge and understanding of cell biology, neither can accurately recapitulate human tissue physiology and its pharmacological response to drugs. For example, up to 50% of compounds eliciting liver injury in man do not show similar effects in animal studies [1]. Increasing awareness of these deficiencies has led to the development of a range of three-dimensional (3D) cell culture models of human tissues, which include unicellular and multicellular models based on known cellular compositions of particular human tissues, and tissue stem cell-derived organoids, collectively termed here micro-tissues. These have enormous potential for helping to elucidate human physiology, mechanisms of disease and their safe treatment [2]. Exploitation of the 3D models is limited by several major challenges. Drug discovery, cell therapy and personalized medicine applications require suites of new technologies that replicate biophysical cell growth conditions, enhance the reproduciblity of micro-tissue handling and, importantly, provide analytical options that capture the complexity of the cellular structures that can now be generated. This requires sustained interdisciplinary collaborations and innovations in the physical sciences. Indeed, many routine research methods do not easily translate from two dimensions to three.This theme issue showcases current research which aims to tackle some of the challenges associated with developing 3D tissues. The contributions come from members of a Medical Research Council (MRC) funded network, 3DBioNet, which aims to establish multidisciplinary teams of scientists from industry and academia who together possess the diverse skills needed to develop 3D micro-tissues. As explained below, the six featured articles span mathematical and computational modelling [3], bioreactor design [4], organoid development [5] and image acquisition and analysis [6].Several papers in this theme issue are devoted to perfusion bioreactors, recognizing that flow bioreactors provide a more realistic environment within which to study cells. For example, in [7], attention focuses on the use of perfusion bioreactors to assess the chemical safety and efficacy of drugs. The authors develop a mathematical/computational model that characterizes the fluid flow and solute transport in a perfusion bioreactor with a single inlet and single outlet. Numerical simulations are used to derive simple relationships between the solute concentration and shear stress experienced by cells seeded in the bioreactor and operating conditions, such as the input flow rate, and the manner in which the cells uptake the solute. In this way, Hyndman et al.[7] use their computational model to identify operating conditions that should be avoided because they would be deleterious to the cells. The computational model is then specialized to describe fluid flow and solute transport ...

show abstract

‘Cloudbuster’: a Python-based open source application for three-dimensional reconstruction and quantification of stacked biological imaging samples

et al. 2022

Self Cite

View full text Add to dashboard Cite

Three-dimensional (3D) spheroid cultures are generating increasing interest in cancer research, e.g. for the evaluation of pharmacological effects of novel small molecule inhibitors. This is mainly due to the fact that such 3D structures reflect physiological characteristics of tumours and the cellular microenvironments they reside in more faithfully than two-dimensional (2D) cell cultures; in addition, they allow the reduction of animal experiments while providing significantly relevant human-based models. Quantification of such organoid structures as well as the mainly slice-based acquisition and thus forced 2D representation of 3D spheroids provide a challenge for the interpretation of the associated generated data. Here, we provide a novel open-source workflow to reconstruct a 3D entity from slice-recorded microscopical images with or without treatment with anti-migratory small molecule inhibitors. This reconstruction produces distinct point clouds as basis for subsequent comparison of basic readout parameters using average computer processor, memory and graphics resources within an acceptable time frame. We were able to validate the usefulness of this workflow using 3D data generated by various imaging techniques, including z -stacks from confocal microscopy and histochemically labelled spheroid sectioning, and demonstrate the possibility to accurately characterize inhibitor effects in great detail.

show abstract

A novel workflow for three-dimensional analysis of tumour cell migration

Cited by 5 publications

References 10 publications

Composition of receptor tyrosine kinase-mediated lipid micro-domains controlled by adaptor protein interaction

Composition of receptor tyrosine kinase-mediated lipid micro-domains controlled by adaptor protein interaction

Three-dimensional biological cultures and organoids

‘Cloudbuster’: a Python-based open source application for three-dimensional reconstruction and quantification of stacked biological imaging samples

Contact Info

Product

Resources

About