Multiscale Molecular Imaging of Immune Cell Variety in the Malignant Microenvironment in Premetastatic Tissue

Metastasis, not primary tumours are the major threat in malignant disease and account for a significant reduction of patient prognosis. At multiple steps during cancer development and metastatic spread, immune cells seem to play a crucial role. It has been shown that immune cells have the potential to promote tumour growth and invasion in tissue, locally adjacent to the primary tumour. For the process of metastasis - though vital - the interactions between tumour- and host cells have not been finally elucidated. It is hypothesised that primary tumours secret a bundle of factors (tumour secreted factors - TSF) which induce an inflammation resembling state in distant, basically healthy tissue. The immune cells, gathering in response to this local inflammation are supposed to enable circulating tumour cells to locally leave the blood stream and seed into the prepared so-called premetastatic niche (PN) to finally form solid metastasis.
Several basic aspects of the concept of PN are still uncharted, as for example the cellular composition of the niche or regulatory mechanisms during PN installment. During this project, we will assess the PN and the contributing immune cells with multiscale target-specific imaging approaches.
As a sensitive marker for immune cell activity and a factor, discussed to be initially involved in establishment of the PN, S100A8/A9 will be the first and exemplary target for our imaging approaches. S100A8/A9 is expressed activity-dependant by specific subsets of immune cells - monocytes during differentiation into macrophages - and has been shown to be present in e.g. lung tissue of tumour bearing animals.
Specific visualisation of this protein will allow for detection of the inflammation in premetastatic tissue of tumour bearing organism and to visualise for the first time whether the effects of TSF are site specific or more generalised. Follow-up examinations will then reveal whether this inflammation is really likely to precede tumour cell invasion and metastatic growth. Modulation of immune cell recruitment will be performed to examine influence on development of metastasis and validation of our results as well as potential therapeutic impact.
Successfully visualised areas of premetastatic inflammation (the PN) will be analysed for their cellular composition. Guided by fluorescence microscopy, macrophages will be isolated from the PN. Analysed for their specific characteristics as compared to macrophages from regular inflammatory reactions, potential targets for genetic intervention ("knock-down") will be identified as those techniques would help to select targets for therapy and diagnostics and basically help to understand the mechanisms, underlying metastatic spread.
The evaluation of these potential target structures and genes and the visualisation of the effects on tumour cell distribution in the organism will keep a next generation of scientists busy. The insights into regulation of tumour cell spread might though alter oncology fundamentally - successful inhibition of metastatic spread would transform cancer from a leading killer in western civilisations to a chronic - controllable - disease.

Aims and objectives
To assess the hypothesis of premetastatic tissue priming and the role of immune cells and associated chemokines during tumour cell migration and seeding.
Methodology
A syngeneic mouse model for breast cancer metastasis will be established using the cell line 4T1, known to preferentially metastasise to the lungs. As control, non- and low-metastatic 4T1 clones will be used, all equipped with a fusion protein allowing for both, fluorescence and radionuclide mediated imaging. A labelled antibody will serve for parallel visualisation of monocyte activity indicating S100A9 expression. For visualisation of cell interactions and the influence of S100A9 on premetastatic niche (PN) formation, intravital and ex-vivo microscopy (incl. FRET, FLIM) will be performed. This will inter alia allow for the first in-vivo visualisation of the aggregation of S100A8 and A9 to form the active S100A8/A9 heterodimer and evaluation of the influence of mon- and dimers on the malignant microenvironment. PN tissue will be identified by signs of inflammation (S100A9) without tumour cell associated signals in whole-body imaging and further examined ("optical biopsy"). PN-associated macrophages will be characterised and transferred to transcriptome profiling, screening for potential target genes for intervention.
Scientific and medical opportunities
This study will result in a model system for further study of the metastatic process in general. As a paradigm, the impact and regulation of S100A8/A9, discussed as a major promoter of the PN, will be evaluated. This and the identification of PN-associated macrophages with regards to their specific makeup and role within this process might reveal novel opportunities to impair tumour metastasis.

Cancer is one of the leading causes of death in western society. Though, the life-shortening aspect of malignant disease is frequently not the primary tumour but the distant metastasis. Even years after successful local treatment of the primary, metastasis may occur.
To elucidate the process of metastasis may lead to more efficient, effective therapy of patients beyond the stage of locally restricted, solitary disease.
It is widely agreed that cure of cancer in the common sense cannot be achieved. Understanding the process of metastasis might - once key substrates of this process have been identified and successfully targeted for therapeutic intervention - though lead to control of the disease in the way of a chronic disorder. This has been identified as one of the major aims of cancer research and this study will contribute towards this aim. The whole process will though take a considerable amount of time.
This study will - potentially - identify targets for intervention. Synthesis of targeted drugs, their testing and validation will still require years after completion of my research project.
Nevertheless it is one of my major goals to contribute to this process which will be of high benefit for virtually all cancer patients. Identification and commercialisation of potential target structures for intervention might be a benefit for the pharmaceutical industry, which will be involved in the evaluation of such targets in an early stage via the King's College London Business Centre.