The Endothelin axis in the primary tumour, the tumour microenvironment and the premetastatic niche – Visualization by target-specific imaging

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The prognosis of malignant disease is mainly determined by the individual potential of the tumour to spread systemically and form distant metastases. The endothelin (ET) axis, composed of three peptide hormones (ET-1, -2 and -3) and the two receptor subtypes ETAR and ETBR, is a potent regulator of immune cell recruitment and activation, putatively involved in the induction of tumour-associated immune cells and premetastatic tissue priming. In recent years, activation of intrinsic immune defence mechanisms for combatting tumour growth and spread and the inhibition of tumour-supportive immune signalling have been established as a promising therapeutic option. The formation of distant metastases by seeding of tumour cells within a fostered premetastatic niche is also a process putatively dependent on this interaction.To visualize the influence of the endothelin axis on immune response within the tumour and the tumour microenvironment is the aim of this study.For the proposed project, the syngeneic murine 4T1 breast cancer model system will be used. The different tumours are well characterised for development, kinetics and immune interaction; they share the genetic background of a spontaneous Balb/c tumour but differ in malignant potential. For visualisation of ETBR, a new small molecular fluorescent probe with high specificity for ETBR will be developed and combined with a recently established probe for ETAR for simultaneous imaging of both receptors. The use in planar 2D and tomographic optical fluorescence imaging as well as multispectral optoacoustic tomography (MSOT) will provide high resolution images of probe distribution in primary tumour and sites of (future) metastasis. The correlative cellular composition of tumour, tumour microenvironment and possible metastases will be analysed using flow cytometry, enabling the differentiation of pro- and anti-inflammatory macrophages, regulatory T cells and natural killer cells. Using this experimental regimen of imaging and consecutive analysis of the cellular representation, we will assess potential effects of ETA/ETB blockade on tumour development and tumour microenvironment makeup. Moreover, the effects of specific blockade of either receptor on the other one will be analysed and visualized.This project will provide a tool for specific and differentiated analysis of the ETA/ETB signaling system; the experiments will elucidate the effects of specific intervention into the endothelin axis and enable evaluation of the therapeutic potential.