MICA: Whole body 3-D imaging of cancer and inflammation in live zebrafish using optical tomography and fluorescence lifetime readouts of signalling

Our overarching aim is to develop a new optical technology platform to directly image mechanisms of disease throughout whole live organisms as they progress over time with a view to improving our understanding of" global" responses to disease progression, and to specific treatments, in order to develop new therapies. To visualise internal processes requires transparent organisms. Zebrafish embryos are particularly interesting because they are inherently transparent and are vertebrates - and so represent better human biology than other transparent organisms. They are increasingly used for biomedical research, with cellular processes being studied using microscopes, and many zebrafish disease models have been produced, including for cancer and inflammation. Unfortunately the use of embryos precludes full studies of disease progression such as tumour development or chronic inflammation and it would be useful to be able to image such processes in more mature fish. Imaging larger fish, however, is challenged by the onset of absorption and scattering of light and the lack of instrumentation for high resolution 3-D imaging through cm scale samples. We would work with non-pigmented mutations of zebrafish and develop new techniques to counter light scattering that we would implement in a novel tomographic imaging system to enable detailed 3-D imaging of changes in tissue structures, cell migration and signalling processes throughout whole juvenile and adult live zebrafish.
Such "global" 3-D imaging is important for cancer because tumour cells typically spread from their original site to other organs. This is called metastasis, which is very difficult to treat and often leads to death. Observing the growth and spread of tumour cells throughout an organism and how they grow at new sites is not possible using the mammalian disease models such as mice that are often used to study cancer biology because they are not optically accessible. Global 3-D imaging is also important for inflammation to study how the response to pollutants spreads throughout an organism, leading to chronic inflammation, and how the body's defences recruit immune cells. However, even with non-pigmented zebrafish, the established optical imaging techniques are restricted to image near the surface and are strongly compromised by optical scattering. Our approach would build on a method called "optical projection tomography" that is similar to x-ray tomography but works with visible light. We would combine it with novel image reconstruction software that builds on the basic physics of light propagation and advanced techniques to exploit characteristic properties of the scattered light. We will apply this novel imaging platform to the study of cancer and inflammation using genetically modified adult zebrafish. To study cancer, we would breed zebrafish in which we can either induce tumours with a chemical reagent or in which we can transplant zebrafish-derived tumours. These cancer models resemble the disease state in humans. To study inflammation, we would use adult zebrafish models that have an analogous immune response to humans and we would study the progression of inflammation resulting from chemical pollutants including cigarette smoke. The various zebrafish models would also be genetically engineered such that specific proteins found in tumour cells, immune cells or blood vessels would be labelled with fluorescent molecules to facilitate imaging of the disease-related structural changes and cell migration. We would also use fluorescently labelled "biosensors" that change their fluorescence properties when specific molecular interactions take place as part of the signalling processes that determine normal cellular function and which become dysregulated in cancer and inflammation. By observing when and where signalling processes are activated or otherwise, we can study the effects of potential therapies such as anti-cancer or anti-inflammatory drugs.

This project aims to develop and apply a new optical tomographic imaging platform to directly observe the progression of disease, including quantitative assays of morphological changes, physiological and molecular signalling processes in live whole juvenile and adult zebrafish that benefit from fully developed vasculature and immune system. We aim to extend optical projection tomography (OPT) to rapid imaging of cell migration using a novel (patent filed) configuration and realise 3-D mapping of optical absorption and fluorescence signals (including fluorescence lifetime imaging - FLIM) in cm-scale non-pigmented zebrafish lines using novel computational reconstruction algorithms to account for weakly scattered light propagation and ballistic light techniques to improve 3-D image quality. We will develop this technology in the context of its application to disease models for both cancer and inflammation. For this we will perform crosses and modify already available zebrafish strains, with fluorescently labelled vasculature and cell types (tumour cells, neutrophils and macrophages). For cancer we would make 3-D measurements of developing (inducible, syngeneic) tumours and the surrounding vasculature and map the localisation and size of resulting metastases over time. We would also read out the activation of Src, MT1-MMP & Rac biosensors using FLIM FRET and correlate changes in these important signalling pathways with tumour development. For inflammation we would image the kinetics of recruitment of inflammatory cell populations throughout a single fish in response to chemical pollutants and correlate this with the activation of signalling networks (Nalp3 inflammasome, TLR) using sensors for cathepsin B, caspase 1 and NF-kB. We would thereby study how chemically induced inflammation varies between larval, juvenile and adult zebrafish. As a basis for future projects, we would start exploring the inflammatory response in tumour bearing zebrafish.

Our local strategic intent is to build on our experience imaging disease models and developing FLIM and FRET technology to establish novel capabilities for global 3-D imaging of biological processes in live organisms such as juvenile/adult zebrafish that would provide an unprecedented resource for disease/drug studies and developmental biology. Once proven, we would make it available via collaboration to academe and industry and ultimately establish a user-contribution-to-costs model to share and sustain the facility, as well as exploring access to industry on a commercial basis. We would also work to make the proposed new imaging technology more widely available to industry and other users through its commercialisation. This would hopefuly benefit academic, clinical and industrial researchers who could buy the commercial instruments and would stimulate the development of competitive techniques, which would benefit the instrumentation sector - particularly companies serving the pharmaceutical and biomedical research communities.

Malignant and chronic inflammatory diseases remain areas of major unmet need and commercial opportunity for the pharmaceutical industry. The capabilities of the proposed instrumentation to image global responses to gene knockdowns or therapeutic interventions in live organisms and to correlate bulk physical changes with changes in the activation of cell signalling pathways would be important for pharmacology and drug testing. It could increase the efficiency of the drug discovery pipeline, e.g. by reducing the time to failure for unsuccessful drug candidates and increasing the opportunities for off-target discovery. The new capabilities to image cancer processes in vivo should benefit the drug discovery industry, providing new tools and information concerning mechanisms of cancer development and progression, and clinical oncologists, who could increase their understanding of cancer metastasis and the value of different therapeutic approaches. The enhanced abilities to image tumour-vasculature interactions, tumour progression and metastasis could increase understanding of the behaviour of tumour cell subpopulations in relation to therapeutic targeting. Along with researchers and clinicians, drug companies could benefit from the improved capability to evaluate the efficacy of drug compounds to slow down or prevent tumour progression and metastasis afforded by imaging whole organisms in vivo and from the identification of novel therapeutic targets. Most importantly, cancer patients could benefit in terms of improved understanding of their disease and increased opportunities to identify effective pharmacological therapies and to develop new ones. In the longer term, this could impact on the development of preventative and therapeutic strategies for cancer that would benefit policy makers and health care providers across the world and could ultimately help reduce the economic burden and human suffering caused by this disease. Beyond cancer, the capability to track the spatio-temporal behaviour of leukocytes and signalling in the context of entire organisms would provide a currently unique resource for understanding the basic physiology of immunity and inflammation as well as disease processes. It could enable genetic or drug screens to understand at a mechanistic level processes that underlie and/or are involved in an extremely broad range of pathologies including cardiac disease, arthritis and obesity and so could impact a very wide range of pharmaceutical researchers, clinicians and patients. Beyond medicine the imaging techniques could be applied to plants and animals to impact food security.

The knowledge and skills gained by the staff working on this project would directly benefit them in terms of increasing their potential for research impact and future employment and could benefit the biophotonics instrumentation and medical research communities who could employ them.