Final Report Summary - 3DMIG (Use of high resolution imaging to elucidate cell migration dynamics within 3D environments)
Project context
The aim of this project is to gain insights into the process of migration in a 3D environment that closely resembles physiological surroundings. The first year of the project involved setting up the collagen gel experiments so that it enabled imaging at comparable resolutions to 2D imaging. This was done by using glass-bottom dishes to set the gels on, and imaging was conducted with either spinning-disk or laser-scanning confocal microscopes. With this set-up, imaging close to the maximum theoretical resolution of the objective could be achieved. Basic characterisation of integrins, adhesion components and actin was carried out using immunofluorescent staining of HEp-3 human epidermoid carcinoma and M21 melanoma cells in 2D and 3D. After these initial studies, the system was optimised for live-cell imaging. To do this, the effects of varying concentration of collagen on migration were assessed and cells were transfected with EGFP-tagged genes relating to cell adhesion complexes, e.g. paxillin, and the cytoskeleton, e.g. F-actin.
Analysis of the cells was predominately done using image analysis software, such as BioImageXD. By collaborating with the BioImageXD development team at University of Turku, Finland, specific analysis functions relative to 3D migration were incorporated in the software. Also, I have been in contact with Royal Institute of Technology, Stockholm, which develops microfluidic chambers for 3D imaging, to discuss how to improve the technology. The goals of the first years work plan were achieved. However, initial testing of the microfluidic device highlighted technical difficulties so it was not possible to use the microfluidic device for the studies proposed in the second year.
Project objectives
The aim of this part of the study was to assess the molecular mechanisms that are regulating 3D migration. Technically, it was hard to consistently visualise focal adhesions in live cell 3D environments. Therefore, it was not possible to conduct dynamic analysis of CMACs such as the FRAP analysis studies on CMAC components. Also, due to previously mentioned technical issues with the microfluidic device, these experiments were stopped. However, rudimentary characterisation of CMAC size and shape was conducted in Hep-3 cells, as M21 did not show substantially in the 3D assays tested that these cells were no longer used. CMAC components were also tested for their effect on migration, e.g. PAK4 and Kindlin-2. Their effects were not significant in the cell lines tested. The inhibition of myosin II had a much larger effect on cell migration, with a reduction in migration observed. Likewise, the results obtained when modifying Rho/Rac GTPases activity was more evident, so technology was developed to look more in-depth into these molecules. Importantly, breakthroughs in biosensor technology has allowed for the activation state of the GTPases RhoA and Rac1 to be measured in live cells. Rac and Rho are considered integral parts of the cells protrusive and contractile signalling pathways respectively. The interplay between these two pathways determines how cells move. A detailed analysis of the Rho and Rac signalling events is planned using these technologies.
To complete the final aim of investigating migratory signalling in vivo, we are collaborating with the lab of Prof. Peter Friedl, Radboud University, Nijmegen, in the Netherlands, who is a leading authority on 3D migration and intravital mouse imaging. H1299 cells incorporating the genes for Rho or Rac activity sensors have been sent to be used in their mouse models.
Project outcomes
Given the importance of cell migration in vital physiological processes like development, wound healing, tumour angiogenesis and metastasis, it is fundamentally important to extend experiments into 3D environments in order to properly understand the human physiology and pathophysiology. Overall, this study has started to gain insight into the processes involved in 3D migration, characterised a number of different cancer cell migration patterns, confirmed the presence of focal adhesions in migrating cancer cells, identified key molecular events (Rho, Rac, Myosin II) governing 3D migration, and developed image analysis technologies. This information is important in understanding of the processes involved in disease states such as cancer metastasis and hopefully will help in the development of treatment strategies for migration-related diseases. Work carried out during this project has also increased the technological capabilities for imaging-based experiments in 3D, with software development and visualisation of signalling events in 3D.
Lastly, an important feature of the international incoming fellowship is the transfer of knowledge to contribute to the expertise of European science. I have collaborated with a number of labs in the EU, (University of Turku, Finland; KTH, Stockholm, Sweden; Centro de Investigaciones Biológicas, Madrid, Spain and Radboud University, Nijmegen, the Netherlands) as well as participating in the EU-FP7 networks Metafight and Systems Microscope Network of Excellence. I have supervised a Masters student, given lectures highlighting some of the studies being undertaken, and collaborated with scientists from Spain on the 3D techniques.
The aim of this project is to gain insights into the process of migration in a 3D environment that closely resembles physiological surroundings. The first year of the project involved setting up the collagen gel experiments so that it enabled imaging at comparable resolutions to 2D imaging. This was done by using glass-bottom dishes to set the gels on, and imaging was conducted with either spinning-disk or laser-scanning confocal microscopes. With this set-up, imaging close to the maximum theoretical resolution of the objective could be achieved. Basic characterisation of integrins, adhesion components and actin was carried out using immunofluorescent staining of HEp-3 human epidermoid carcinoma and M21 melanoma cells in 2D and 3D. After these initial studies, the system was optimised for live-cell imaging. To do this, the effects of varying concentration of collagen on migration were assessed and cells were transfected with EGFP-tagged genes relating to cell adhesion complexes, e.g. paxillin, and the cytoskeleton, e.g. F-actin.
Analysis of the cells was predominately done using image analysis software, such as BioImageXD. By collaborating with the BioImageXD development team at University of Turku, Finland, specific analysis functions relative to 3D migration were incorporated in the software. Also, I have been in contact with Royal Institute of Technology, Stockholm, which develops microfluidic chambers for 3D imaging, to discuss how to improve the technology. The goals of the first years work plan were achieved. However, initial testing of the microfluidic device highlighted technical difficulties so it was not possible to use the microfluidic device for the studies proposed in the second year.
Project objectives
The aim of this part of the study was to assess the molecular mechanisms that are regulating 3D migration. Technically, it was hard to consistently visualise focal adhesions in live cell 3D environments. Therefore, it was not possible to conduct dynamic analysis of CMACs such as the FRAP analysis studies on CMAC components. Also, due to previously mentioned technical issues with the microfluidic device, these experiments were stopped. However, rudimentary characterisation of CMAC size and shape was conducted in Hep-3 cells, as M21 did not show substantially in the 3D assays tested that these cells were no longer used. CMAC components were also tested for their effect on migration, e.g. PAK4 and Kindlin-2. Their effects were not significant in the cell lines tested. The inhibition of myosin II had a much larger effect on cell migration, with a reduction in migration observed. Likewise, the results obtained when modifying Rho/Rac GTPases activity was more evident, so technology was developed to look more in-depth into these molecules. Importantly, breakthroughs in biosensor technology has allowed for the activation state of the GTPases RhoA and Rac1 to be measured in live cells. Rac and Rho are considered integral parts of the cells protrusive and contractile signalling pathways respectively. The interplay between these two pathways determines how cells move. A detailed analysis of the Rho and Rac signalling events is planned using these technologies.
To complete the final aim of investigating migratory signalling in vivo, we are collaborating with the lab of Prof. Peter Friedl, Radboud University, Nijmegen, in the Netherlands, who is a leading authority on 3D migration and intravital mouse imaging. H1299 cells incorporating the genes for Rho or Rac activity sensors have been sent to be used in their mouse models.
Project outcomes
Given the importance of cell migration in vital physiological processes like development, wound healing, tumour angiogenesis and metastasis, it is fundamentally important to extend experiments into 3D environments in order to properly understand the human physiology and pathophysiology. Overall, this study has started to gain insight into the processes involved in 3D migration, characterised a number of different cancer cell migration patterns, confirmed the presence of focal adhesions in migrating cancer cells, identified key molecular events (Rho, Rac, Myosin II) governing 3D migration, and developed image analysis technologies. This information is important in understanding of the processes involved in disease states such as cancer metastasis and hopefully will help in the development of treatment strategies for migration-related diseases. Work carried out during this project has also increased the technological capabilities for imaging-based experiments in 3D, with software development and visualisation of signalling events in 3D.
Lastly, an important feature of the international incoming fellowship is the transfer of knowledge to contribute to the expertise of European science. I have collaborated with a number of labs in the EU, (University of Turku, Finland; KTH, Stockholm, Sweden; Centro de Investigaciones Biológicas, Madrid, Spain and Radboud University, Nijmegen, the Netherlands) as well as participating in the EU-FP7 networks Metafight and Systems Microscope Network of Excellence. I have supervised a Masters student, given lectures highlighting some of the studies being undertaken, and collaborated with scientists from Spain on the 3D techniques.