Experiencing the micro-world - a cell's perspective

Lead Research Organisation: University of Nottingham
Department Name: Faculty of Engineering

Abstract

In the body, most cells grow in close contact with other neighbouring cells and with a local matrix of proteins and sugars that combine to provide an instructive microenvironment. Until recently, most research labs (in both academic and industrial settings) have used 2D cultures of cells on plastic to study cell behaviour, a significant departure from what is actually happening in vivo that can limit the applicability of their research. However, there has been a recent and dramatic shift away from traditional 2D culture to the use of complex, 3D cultures, that more effectively mimic the micro-environment experienced by cells in vivo. This development impacts directly on fields such as regenerative medicine, drug discovery and cancer research, with significant opportunities for improved in vitro modelling of cell behaviour. Despite these improvements in culture techniques, the interaction of the cells with their local microenvironment - a key target in therapies for cancer, wound healing, and fibrosis etc. - remains a 'black box' with technologies unable investigate these environments at the cell level. This proposal will 'open that box', developing the technology and methodology urgently required to fully explore 3D cell cultures on length scales comparable, or smaller than, single cells.

The currently accepted protocol to characterise natural and synthetic matrices, uses a bulk rheometer to produce a single, averaged value of the viscosity and elasticity of the material, destroying the sample in the process. Information about the matrix local to the cells growing inside the samples is lost. Our vision is to image and characterise 3D cell culture environments in all three spatial dimensions, over an extended time course, and on a single multifunctional instrument so that the information can be integrated and mapped. To achieve this we will develop a minimally-invasive technique to measure the 3D micro-rheology of the extracellular matrix using nano- (smaller than the cells) and micro-sized (can be the same size at the cells) beads as local probes. These probes will be held at a fixed position within the matrix using an optical trap and their Brownian motion in all three spatial dimensions tracked using multiplane imaging. The micro-rheology (viscosity and elasticity) of the extracellular matrix local to the probe is extracted from temporal analysis of the Brownian motion. To achieve deep 4D (x,y,z, time) images of live 3D cell cultures, we will combine light sheet microscopy with adaptive optics (a technique for correcting for sample aberrations that reduce image quality deep into complex samples). The final multifunctional platform will be the exciting culmination of these 4 microscopy techniques - optical trapping, multiplane imaging, light sheet microscopy and adaptive optics - capable of imaging and micro-mechanically sensing the 3D environment close to cells.

The output from this work will be the innovation required to allow scientists to study how cells interact with their local microenvironment, combining technologies in a way that's not been possible previously, to observe both the cells, and the forces they exert and are responding to, as they grow and move in 3D space over time. The ability to study cell behaviour in this way is of importance for developing therapies for diseases where cells respond abnormally to signals from their local matrix, such as cancer, providing targets for new drug design. We will include a demonstration of how this can work in our study using both traditional anti-cancer drugs and more innovative therapies such as functionalised nanoparticles. We anticipate that the technology will be useful to both academics and industry (particularly drug discovery in the pharmaceutical industry) and we will work closely with these groups throughout the course of this project to ensure that, once proven, this technology can work for them.

Planned Impact

We anticipate that the technology we will develop will synergise with the recent shift from 2D cell culture models by adding significant value to the use of in vitro 3D cell culture model systems. The route to impact here is two-fold, with the new technology aiding the design of new matrices that better replicate the in vivo microenvironment (with the potential to personalize therapeutic screening) and enabling the effective monitoring of cell and matrix responses to potential new therapeutic candidates. A better understanding of how cancer cells respond to drugs and delivery systems is of clear value to those developing new treatments for many diseases and disorders. As highlighted in a recent high-impact review 'Targeting the extracellular matrix (ECM), the enzymes that remodel it and the receptors that transduce their signals offers promising therapeutic opportunities for many diseases' [1]. By providing a new way of probing cell-matrix interactions, from the cell's perspective, we aim to open up opportunities to develop novel therapeutics with immediate relevance to cancer and fibrosis where druggable targets involved in cell-matrix interactions have already been identified. We recognise that new drugs and formulations take many years and vast budgets to develop. Improved methods for early stage screening to identify lead candidates and exclude poor performers (fail early/fail cheap) early is of critical significance.

Industry impact can be further appreciated in economic and social terms, via the recent Association of British Pharmaceutical Industry (ABPI) document on 'Bridging the skills gap in the biopharmaceutical industry' 2015. This ABPI document emphasized the importance of pharmaceutical formulation as a critical discipline, with a survey of the pharmacy sector, showing that formulation is a top priority area, with 50% of respondents' classifying formulation it as 'high priority'. In addition, the document highlighted significant concerns (>60% of the respondents) to recruit an experienced work force in pharmaceutical formulation. In line with these findings AstraZeneca-MedImmune recently launched a postgraduate programme that illustrates the need for pharmaceutical formulation scientists to work in industry driven research [2]. Our proposed project will train PDRAs, technical staff (and associated PhDs) in areas critical for the pharmaceutical industry. The development of a formulation screening system suitable for in vitro 3D cell assays, and researchers trained in how to apply it, as set out here would be very valuable to industry, as noted in e-mail correspondence with Dr Delyan Ivanov (AstraZeneca).

This project would also fits well with the move from animal-based models (e.g. patient derived xenografts, commonly used in breast cancer research) towards better defined, better controlled in vitro model systems that aim to improve on the poor rate of translation of highly-cited animal studies to human trials (less than one third [3]). The impact here will be in improved drug screening platforms both at the basic science level (University researchers, R&D labs in industry) and at the drug development level (mostly small/large Pharma), with patients and society benefiting from development of improved drugs for what can be long-lasting, debilitating and costly diseases.

[1] Remodeling the extracellular matrix in development and disease. (2014) Bonnans C. et al. Nat. Rev. Mol. Cell Biol. 15, 786-801.
[2] https://careers.astrazeneca.com/students/programmes/pharmaceutical-technol.
[3] Lost in translation: animal models and clinical trials in cancer treatment. (2014) Mak et al. Am. J. Trans. Res. 15;6(2),114-8.

Publications

10 25 50
 
Description We now have the completed optical instrument at the University of Nottingham which was one of the aim aims for this project. The instrument can image samples of cell clusters in 3D over long-time courses and map to these images the local 3D mechanical properties of the material surrounding the cells. Some of the instruments capabilities include the ability to image live cells in situ whilst they are growing, image deep into a sample of cells (~400 microns) and to study the micro-rheology of locally varying samples. We have shown this instrument to be useful for studying plankton systems in a new collaboration with the University of Lincoln and are currently using it to investigate breast cancer cell clusters and nanoparticle drug delivery.
Exploitation Route We have applied for NERC funding to continue the off-shoot project looking the local rheology surrounding plankton systems. This is an exciting new avenue which could make an important contribution in understanding the eco-system of the oceans.
Sectors Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://nu-sense.ac.uk/home.aspx
 
Description Fully humanised 3D vascular perfused model for breast cancer modelling and therapeutic screening
Amount £75,911 (GBP)
Funding ID NC/T001259/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2019 
End 09/2021
 
Description Lighting the Way to a Healthy Nation - Optical 'X-rays' for Walk Through Diagnosis & Therapy
Amount £5,577,754 (GBP)
Funding ID EP/T020997/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 06/2020 
End 05/2025
 
Description Replacing the need for patient-derived xenografts and matrigel organoid culture as preclinical models for breast cancer
Amount £75,591 (GBP)
Funding ID NC/T001267/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2019 
End 06/2020
 
Description U-care: Deep ultraviolet light therapies
Amount £6,132,366 (GBP)
Funding ID EP/T020903/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 06/2020 
End 06/2025
 
Description Understanding How Matrix Remodelling & Respiratory Infection Impact Progression Of Idiopathic Pulmonary Fibrosis Using Stem Cell Derived Alveolar Cells
Amount £93,000 (GBP)
Organisation British Lung Foundation (BLF) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2019 
End 08/2022
 
Title Peptide gels for 3D breast cancer models 
Description Current materials used for in vitro disease models are often limited by their poor similarity to human tissue, batch-to-batch variability and high complexity in composition and manufacture. We have developed a "blank slate" culture environment that can be customized by incorporating matrix components specifically selected to match the target tissue, with mechanical properties controlled independently and simultaneously. Based on a self-assembling peptide hydrogel, this system contains no exogenous proteins or glycosaminoglycans: only those specifically added, or those synthesized by the cells in culture. This 3D culture platform therefore provides full control over biochemical and physical properties, allowing the composition and mechanics of the tissue of interest to be recapitulated in vitro. As proof-of-concept, we designed a panel of hydrogels designed to mimic the stages of breast cancer progression. Controlling the peptide gelator concentration allows hydrogel stiffness to be matched to normal breast (<1 kPa) or breast tumour (>1 kPa), with higher stiffness favouring the viability of breast cancer cells over normal breast cells. In parallel, these hydrogels may be modified with matrix components relevant to human breast, such as collagen I and hyaluronan. The choice and concentration of these additions control the size, shape and organisation of the breast epithelial cell structures formed in co-culture with fibroblasts. This system therefore provides a means of unravelling the individual influences of matrix, mechanical properties and cell-cell interactions in cancer and disease. 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2018 
Provided To Others? Yes  
Impact We have presented the gel technology at events specifically designed to encourage uptake by other researchers working in this area (e.g. BACR meeting in Leeds, May 2018) and have trained researchers from other groups to encourage uptake of the technology. These training visits were supported financially by NC3Rs. We have also developed additional collaborations related to this technology including with a commercial partner able to provide the raw materials required for the gels. By demonstrating the applicability of the peptide gels to breast cancer modelling we have also attracted additional collaborations with groups working on fibrosis and the support of stem cell differentiation. 
 
Description Adelaide Nottingham Joint PhD Studentship 
Organisation University of Adelaide
Country Australia 
Sector Academic/University 
PI Contribution A new joint PhD student started in January 2021. The student will be supervised by Kylie Dunning and Amanda Wright and will spend years 1 and 3 in Adelaide and year 2 in Nottingham
Collaborator Contribution Initial contact was made via Kylie Dunning and we then applied jointly for this studentship.
Impact PhD studentship
Start Year 2020
 
Description Alexander Thompson - joint PhD student 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint PhD studentship via the EPSRC Regenerative Medicine CDT.
Collaborator Contribution Joint PhD studentship via the EPSRC Regenerative Medicine CDT.
Impact This is a multidisciplinary collaboration with supervisors in Medicine (Merry, Thompson, Arkill) and Engineering (Wright)
Start Year 2017
 
Description Andrew Hook 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution My group provide expertise in glycosaminoglycan structure and function.
Collaborator Contribution Andrew Hook is an expert in surface structural analysis and we are working together to develop methods for the rapid analysis of glycosaminoglycan structures.
Impact Fully funded studentship associated with Andrew's Nottingham Research Fellowship
Start Year 2019
 
Description Joint PhD student with Kishan Dholakia 
Organisation University of St Andrews
Country United Kingdom 
Sector Academic/University 
PI Contribution We have a joint student under the Nottingham and Adelaide Studentship scheme
Collaborator Contribution With Kylie Dunning at the University of Adelaide we were awarded a joint PhD studentship via the Nottingham and Adelaide partnership. Kishan Dholakia is on secondment at the University of Adelaide and is part of the supervisory team. The student started in January 2021.
Impact PhD student started in January 2021
Start Year 2020
 
Description Kenton Arkill - joint PhD student 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We jointly supervised a PhD student supported by the EPSRC/MRC CDT in Regenerative Medicine
Collaborator Contribution We jointly supervised a PhD student supported by the EPSRC/MRC CDT in Regenerative Medicine
Impact The is a multi-disciplinary PhD project with supervisors in Medicine (Thompson, Arkill, Merry) and Engineering (Wright).
Start Year 2017
 
Description Neil Thomas 
Organisation University of Nottingham
Department School of Chemistry Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We have started working with a group based in Chemistry at Nottingham. The group have expertise in chemical functionalisation strategies that we can apply to our peptide gels. We are able to provide a biologically relevant test system in which to demonstrate the utility of their methods.
Collaborator Contribution The chemists have already developed a variety of methods for functionalization of peptide fibres and this technology can be transferred to our peptide gels.
Impact This collaboration links organic chemistry (Neil Thomas) and matrix biology (Cathy Merry). We have recently been awarded a BBSRC iCASE studentship with which to follow up on our preliminary work.
Start Year 2018
 
Description Pepceuticals 
Organisation Pepceuticals
Country United Kingdom 
Sector Private 
PI Contribution We are working with this company, using the peptides they synthesise to create 3D hydrogels for cell encapsulation.
Collaborator Contribution Pepceuticals are a synthetic peptide company and are interested in novel products.
Impact We have recently been awarded a BBSRC iCASE studentship (PI is Neil Thomas, Cathy Merry CoI with Kamal Badiani from Pepceuticals) to create novel functionalised peptides that can be applied to the peptide gel system. This links organic chemistry (Neil Thomas), matrix biology (Cathy Merry) and peptide synthesis at a commercial scale (Kamal Badiani).
Start Year 2018
 
Description Stuart Humphries - Evolution and Ecology Research Group 
Organisation University of Lincoln
Country United Kingdom 
Sector Academic/University 
PI Contribution Our optical trapping micro-rheology technique has been used to assess the local viscosity gradients close to individual phytoplankton and marine aggregates
Collaborator Contribution The group Lincoln have supplied the to samples and preformed equivalent measurements using a multi-particle tracking method for characterising micro-scale viscosity gradients.
Impact This a multidisciplinary collaboration including Manlio Tassieri (Engineering, University of Glasgow), Amanda Wright (Engineering, University of Nottingham) and Stuart Humpheries (Life Sciences, University of Lincoln).
Start Year 2019
 
Description Building materials for a workshop at the EMBO practical course 'Techniques for Mammary Gland Research' 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact We prepared slides for presentation as part of the prestigious EMBO practical course in Techniques for Human Mammary Gland Research, presented by Prof. Maria Vivanco (CIC BioGune. The course ran at EMBL in Heidelberg. The slides were designed to give a general overview of the use of peptide hydrogels as an alternative to the use of animal models of breast biology.
Year(s) Of Engagement Activity 2019
URL https://www.embl.de/training/events/2020/MAM20-01/
 
Description Guardian article - linked to Nobel Prize announcement 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A quote was provided for a Guardian article on the 2018 Nobel Prize for Physics. As a result the article referred directly to the experiencing the micro-world grant.
Year(s) Of Engagement Activity 2018
 
Description NC3Rs/Unilever joint workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact This was a scoping workshop to identify opportunities for NC3Rs to work towards the development of animal product free in vitro systems. This involved a variety of industrial and academic partners looking at various 'needs' where we could/should be developing alternatives to animal use. My participation was focused on non-animal alternatives to Matrigel.
Year(s) Of Engagement Activity 2019
 
Description Nottingham Breast Cancer Awareness Event 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact As part of the Nottingham Breast Cancer Awareness day we organised a 'hands-on' demonstration to provide more information about our use of the peptide gel technology for 3D culture of breast cancer models. We talked with multiple groups of patients, fundraisers and the general public, answering their questions about our research and why we were trying to reduce the numbers of animals used in the lab. Many of the groups said that they hadn't considered this aspect of the work previously.
Year(s) Of Engagement Activity 2018
 
Description Participation in an ENBDC (European Network for Breast Development and Cancer) workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact We participated in a workshop that ran as a satellite event to the main Manchester 2019 ENBDC meeting. We gave an introductory talk then moved the group through to the lab where we gave a 'hands on' presentation demonstrating how to use the hydrogel and addressing questions from the group. The group included major group leaders from European breast biology groups as well as representatives from Industry. In a follow up survey, this group were shown to be currently using over 1700 mice p/a in their studies and all attendees indicated that the peptide hydrogel technology was of interest to them as an alternative method.
Year(s) Of Engagement Activity 2019
URL https://enbdc.org/
 
Description Pint of Science (Nottingham) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact A 30 min presentation as part of the Pint of Science festival
Year(s) Of Engagement Activity 2018
 
Description Wonder 2019 
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 Public/other audiences
Results and Impact We ran a stall at the University of Nottingham's premier outreach event 'Wonder'. We used the stall to highlight two aspects of the group's work. One was the use of peptide hydrogels to create improved models of disease (primarily cancer). The other was the importance of sulphated glycosaminoglycans in health and disease. Both aspects were very popular - we had hands-on exhibits targetted at children and additional information available for older children/adults so we had a lot of engagement and positive feedback. Mostly this was raising the awareness of the importance of the 3Rs in laboratory work and cancer research in particular.
Year(s) Of Engagement Activity 2019
URL https://www.nottingham.ac.uk/wonder/