Creating bespoke functionalised peptide gels to model the brain tumour microenvironment
Lead Research Organisation:
University of Nottingham
Department Name: School of Medicine
Abstract
Introduction
Current in vitro models used to study children's brain cancer typically fail to recapitulate the complexity of the brain tumour microenvironment. 2D monolayer cultures, or 3D models containing animal-derived products, unreliably represent human extracellular matrices (ECM), and raise ethical questions of using animal-derived products in research. Therefore, we aim to optimise a synthetic peptide gel model which can be selectively functionalised with various ECM components to mimic specific tumour microenvironments.
Materials and Methods
The self-assembling FEFEFKFK gelator octapeptide has been used to encapsulate a variety of cell types with excellent viability. Functionalisation of the gel can be achieved by entrapment of soluble ECM components during cell encapsulation or by covalent modification of the gelator peptide. Mouse embryonic stem (mES) cells have been used as a tool for detecting the biological impact of using specific glycosaminoglycans to functionalise the gels.
Results
mES cells grown in the peptide gels can be induced to differentiate in serum free culture. We have previously demonstrated that the glycosaminoglycan heparan sulphate (HS) is essential for mES cell differentiation to the neural lineage. Using mES cells deficient in HS synthesis we can probe the ability of exogenous HS, added in to the gel, to support differentiation. This provides a sensitive and relevant read-out for functionalisation with biologically active ECM components.
Discussion
Initial results demonstrate that cell types relevant to childhood brain tumours can be cultured in the peptide gels. Functionalisation of the gel was found to drive characteristic changes in the behaviour of at least one cell type. We have constructed a test environment to evaluate the biological effect of modifications to the hydrogel. Our aim is to develop a model that recapitulates the brain tumour microenvironment to improve upon current models, without the need for animal-derived products and their associated variability.
Current in vitro models used to study children's brain cancer typically fail to recapitulate the complexity of the brain tumour microenvironment. 2D monolayer cultures, or 3D models containing animal-derived products, unreliably represent human extracellular matrices (ECM), and raise ethical questions of using animal-derived products in research. Therefore, we aim to optimise a synthetic peptide gel model which can be selectively functionalised with various ECM components to mimic specific tumour microenvironments.
Materials and Methods
The self-assembling FEFEFKFK gelator octapeptide has been used to encapsulate a variety of cell types with excellent viability. Functionalisation of the gel can be achieved by entrapment of soluble ECM components during cell encapsulation or by covalent modification of the gelator peptide. Mouse embryonic stem (mES) cells have been used as a tool for detecting the biological impact of using specific glycosaminoglycans to functionalise the gels.
Results
mES cells grown in the peptide gels can be induced to differentiate in serum free culture. We have previously demonstrated that the glycosaminoglycan heparan sulphate (HS) is essential for mES cell differentiation to the neural lineage. Using mES cells deficient in HS synthesis we can probe the ability of exogenous HS, added in to the gel, to support differentiation. This provides a sensitive and relevant read-out for functionalisation with biologically active ECM components.
Discussion
Initial results demonstrate that cell types relevant to childhood brain tumours can be cultured in the peptide gels. Functionalisation of the gel was found to drive characteristic changes in the behaviour of at least one cell type. We have constructed a test environment to evaluate the biological effect of modifications to the hydrogel. Our aim is to develop a model that recapitulates the brain tumour microenvironment to improve upon current models, without the need for animal-derived products and their associated variability.
Organisations
| Description | We have demonstrated the utility of our peptide hydrogel system in a differentiation experiment. Ext1null mouse embryonic stem cells, with lack endogenous heparan sulphate and therefore cannot undergo neural specification, were able to differentiate to neural progenitor cells in gels functionalised with exogenous heparan sulphate or heparin. We have also begun work on a bespoke covalent funtionalisation technique which employs the transpeptidase sortase A. By extending the octapeptide to include an oligogycine tag, we have shown that sortase is able to covalently attach eGFP which has an expressed complimentary LPETG tag. This initial experiment validates this approach and will be used to covalently attach other proteins in the future. Work is also ongoing on using copper-catalyzed azide-alkyne cycloaddition as a potential functionalisation technique but is yet to yield any positive results. |
| Exploitation Route | Further optimisation of functionalisation techniques is still required. When these have been optimised, the creation of new bespoke peptide hydrogel models to model a variety of tissues and organ systems will be possible. This could be put to use by other research scientists in a variety of fields. |
| Sectors | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | University Open Day - Cancer Sciences |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Other audiences |
| Results and Impact | In 2019, I participated in a University of Nottingham Open Day where I helped with guided tours of some of the university's research labs and answered any questions prospective students and their parents had about the new Cancer Sciences undergraduate degree. As a former Oncology masters student at the University of Nottingham I was well equipped to give them insight into what an undergraduate degree in this field would be like here at the university. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Wonder 2019 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | Wonder in an annual event hosted by the University of Nottingham at Highfields Park. It is an opportunity for the local community to come and find out about our groundbreaking research and teaching first hand and to have a go at a huge range of interactive activities. 50+ people visited our stand to find out about the role of glycans in health and disease and how we are using peptide hydrogels to model different tissues for research. |
| Year(s) Of Engagement Activity | 2019 |
| URL | https://www.nottingham.ac.uk/home/featureevents/2019/wonder-2019.aspx |