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

恶性疾病的预后主要取决于肿瘤个体系统性扩散和形成远处转移的潜力。内皮素（ET）轴由三种肽激素（ET-1、-2和-3）和两种受体亚型ETAR和ETBR组成，是免疫细胞募集和激活的有效调节剂，据推测参与肿瘤相关免疫细胞的诱导和转移前组织启动。近年来，激活内在免疫防御机制来对抗肿瘤生长和扩散以及抑制肿瘤支持免疫信号已被确定为一种有前途的治疗选择。通过在培养的转移前生态位内播种肿瘤细胞形成远端转移也是一个推定依赖于这种相互作用的过程。可视化内皮素轴对肿瘤内免疫反应和肿瘤微环境的影响是本研究的目的。本项目将采用同基因小鼠4T1乳腺癌模型系统。不同的肿瘤在发育、动力学和免疫相互作用方面有很好的特征；它们都具有自发性Balb/c肿瘤的遗传背景，但在恶性潜能方面有所不同。为了实现ETBR的可视化，将开发一种新的具有高特异性的ETBR小分子荧光探针，并将其与最近建立的ETAR探针相结合，同时成像两种受体。平面二维和层析光学荧光成像以及多光谱光声断层成像（MSOT）的使用将提供原发肿瘤和（未来）转移部位探针分布的高分辨率图像。利用流式细胞术分析肿瘤的相关细胞组成、肿瘤微环境和可能的转移，使促炎性和抗炎性巨噬细胞、调节性T细胞和自然杀伤细胞分化。利用这种成像和细胞表征连续分析的实验方案，我们将评估ETA/ETB阻断对肿瘤发展和肿瘤微环境构成的潜在影响。此外，特异性阻断任一受体对另一受体的影响将被分析和可视化。该项目将为ETA/ETB信号系统的具体和差异化分析提供工具；该实验将阐明特异性干预内皮素轴的作用，并对其治疗潜力进行评估。

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.