Experiencing the micro-world - a cell's perspective

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.

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.