High content analysis of 3-D cell cultures with multidimensional fluorescence imaging

The response of cells to stimuli depends on cell signalling processes, which are combinations of molecular interactions. Disease is associated with deviations from normal signalling processes and so reading out molecular interactions in cells can help elucidate mechanisms of disease and also provide a means to evaluate potential therapeutic drugs. Many of the signalling molecules in cells are proteins and their interactions are widely studied using microscopy with proteins of interest being labelled with fluorescent molecules ("fluorophores"). For drug discovery, fluorescence imaging of arrays of cells is automated so that the effect of many compounds on cell signalling processes can be "assayed". Fluorophores are "excited" by radiation at a wavelength at which they absorb and the resulting characteristic emission (fluorescence) is recorded using an imaging detector. By labelling different kinds of protein with different fluorophores and comparing the images at their respective emission wavelengths, it is possible to learn about protein interactions by observing when they appear in the same place at the same time. Unfortunately this "co-localisation" is usually limited by diffraction of light to a resolution of a few 100 nm - much larger than the size of typical signalling proteins (~1-10 nm). It is possible, however, to confirm interactions using Förster Resonant Energy transfer (FRET), which entails labelling the proteins with different fluorophores and observing when energy is transferred between them. This energy transfer can only occur if they are within ~10 nm of each other. The most quantitative way to read out interactions using FRET is through the reduction in the fluorescence lifetime of the "donor" fluorophore as it loses energy due to the energy transfer. Fluorescence lifetime measurements can be made in every pixel and this fluorescence lifetime imaging (FLIM) enables protein interactions to be mapped in space and time. Fluorescence lifetime can also be used to distinguish different molecular species or different states of naturally fluorescent molecules that are involved in regulating the consumption of energy in the cell, which can also be altered by disease.

For cell biology research and for drug discovery, fluorescence-based studies typically involve imaging thin - and therefore transparent - layers of cells. Unfortunately this is not a physiologically normal environment for cells and they often behave differently compared to when they are in their normal 3-D tissue context. However, it is highly challenging to image cells in native biological tissue and even more so to realise this in a high throughput mode for drug testing. Instead, there is increasing interest in assaying synthetic 3-D cultures comprising many cells that interact with each other, presenting behaviour reminiscent of that in native tissue with greater optically accessibility - although unfortunately they can scatter and absorb light more strongly than thin layers of cells. Such 3-D cell cultures can be arrayed for rapid imaging, however, and we propose to develop an automated platform to provide 3-D images of such cell cultures, utilising FLIM to read out molecular interactions. For this we will optimise the 3-D cell culture and labelling methodologies for fluorescence imaging and will develop and evaluate automated microscopes for FLIM-based assays. For larger samples we will utilise multiphoton excitation, which entails illuminating the fluorophores at twice the wavelength usually required for excitation, such that they need two photons arriving simultaneously. This two photon absorption is intensity-dependent and so can be arranged to occur only in the focal plane of a scanning laser beam such that the emitted photons all originate from a specific depth in the sample. Scanning the focal plane then enables 3-D imaging. The longer wavelength light is less phototoxic and is scattered less by the sample, thus enabling deeper imaging.

3-D cell cultures are being explored for a range of assays and research studies, including cancer progression and stem cell differentiation, since their signalling processes are expected to be much closer to the in vivo context than for conventional monolayer cell cultures. We aim to develop an automated multidimensional fluorescence imaging platform for assays of signalling processes in 3-D cell cultures, particularly tumour spheroids, including FLIM for quantitating protein interactions and changes in cellular metabolism. Unfortunately, their increased scattering, aberration and attenuation of optical signals make them more challenging to image than 2-D cell cultures. With GSK, we propose to explore and develop high content tools and methodologies for image-based assays of cell signalling and morphological changes in 3-D cell cultures. Building on our experience developing automated FLIM multiwell plate readers, we propose to take advantage of the inherently ratiometric nature of fluorescence lifetime measurements that enables quantitative molecular readouts in spite of scattering and attenuation of excitation and fluorescence emission - as already exploited for intravital imaging. In particular, we would use FLIM to quantify FRET readouts of protein interactions and genetically expressed biosensors as well as autofluorescence of NADH and collagen. We would investigate trade-offs between 3-D cell culture methods, sample preparation, imaging speed and functional readouts for exemplar assays of protein multimerisation, of genetically expressed FRET biosensors and autofluorescent metabolites and of protein-histone association. This project would include spinning disc confocal and multibeam multiphoton microscopy to image spheroids of different sizes and at different speeds, in order to give GSK (and other pharma) insights into optical approaches for assaying 3-D cell cultures.

The proposed technology platform and methodology should benefit researchers wanting to understand cell signalling processes and disease mechanisms and clinicians and pharma wanting to develop therapies since the 3-D cell cultures are believed to resemble "normal" behaviour in vivo more closely than the thin layers of cells cultured on transparent substrates that are mainly used for microscopy in cell biology research and assays for drug discovery. From discussions with our pharma colleagues (GSK and AZ) and at live cell assay meetings (e.g. Informa Life Sciences Drug Discovery Innovations, Berlin 2013; SMi's 6th annual Cell Based Assays, London 2013), there is increasing interest in drug testing with 3-D cell cultures including tumour spheroids, which would specifically benefit cancer studies, and stem cell cultures that can be used for a broad spectrum of applications including regenerative medicine.

Improving the physiological relevance of assays could impact the efficiency of the drug discovery pipeline, perhaps on a 5 year timescale, and ultimately reduce its cost - with further economic and societal benefits. It could also reduce the need for animal testing. Unfortunately, it is not usually considered practical to screen with 3-D cell cultures, partly because their higher absorption and scattering and less reproducible growth compared to monolayers of cells make high content analysis more challenging. While today there are a few vendors selling systems for growing 3-D cell cultures, they are usually imaged with conventional plate readers that provide wide-field images or sectioned images near the surface of, e.g. tumour spheroids and we are not aware of any automated assays of cell signalling in 3-D cell cultures. It is therefore of immediate interest to GSK and other pharma to better understand the potential for using 3-D cell cultures in drug discovery and the most direct impact would come from providing this information to GSK and subsequently to other pharma through presentations at conferences, including pharma industry-orientated meetings where the applicants are often invited to speak. Increasing the efficiency of the drug discovery pipeline would lead to economic benefits (over 5-15 years) for pharma and related industrial sectors. Following the impact on pharma, the beneficiaries of more efficient drug discovery would include patients benefitting from improved therapies and healthcare providers benefitting from cheaper, more effective drugs and fewer therapeutic interventions. This societal impact would be on a timescale of >10 years.

The specific biology applications in the project could impact research across a wide range of diseases including cancer, diseases such as diabetes associated with metabolism and obesity and autoimmune diseases, with concomitant benefits to patients, healthcare providers and industry in the longer term. The ability to read out molecular interactions, metabolic changes and the formation of complex tissue structures in 3-D cell culture could be important for other fields of biomedical research and industry including tissue engineering, regenerative medicine and monitoring of other therapeutic approaches such as radiation therapy. For the technologies and methodologies developed in this project to be accessible by pharma, other industry and academe, they need to be commercially available. As this project highlights important potential applications of 3-D cell cultures, it would present opportunities for instrumentation and software companies to address these potential markets, thereby generating economic impact over a ~3-10 year timescale.

This project would provide world-class training for the RAs in imaging technology and software, cell-based assays and the biology of 3-D cell cultures, all of which are in demand and could help establish careers in academic or commercial research and development as well as more general careers in management, consulting and technical sales.