Characterization of the premetastatic niche at different sites and in vivo imaging of granulocytic myeloid derived suppressor cells

癌症的一个重要标志是出现转移，通常代表癌症疾病的最后阶段。值得注意的是，某些肿瘤类型倾向于转移性扩散的特定器官。与将转移形成描述为随机事件的过时理论相反，转移扩散现在被认为是多步骤过程，在循环肿瘤细胞能够播种之前，由靶器官中的转移前小生境（PMN）的形成启动。作为原发性肿瘤信号传导的结果，发生不同骨髓来源的干细胞类型的浸润、特征性粘附分子的表达增加和细胞外基质的重塑。原发性肿瘤内的缺氧诱导导致PMN出现增加的信号传导，伴随粒细胞髓源性抑制细胞（G-MDSC）的浸润。到目前为止，在最近的出版物中主要描述了肺中PMN的发展。在本项目中，将在小鼠乳腺癌转移模型（PymT）中研究骨髓和脑中PMN的出现。将使用对骨髓和脑转移具有特异性的特别生成的樱桃红/荧光素酶转基因PymT乳腺癌细胞。在这些实验中，用来源于骨髓或脑特异性乳腺癌细胞的缺氧条件培养基处理实验动物，诱导相应部位的PMN出现增加。本项目主要研究骨髓和脑中中性粒细胞的特性，以及G-MDSC向中性粒细胞迁移的体内成像，在资助期间可分为以下子项目：A）骨髓和脑中性粒细胞中G-MDSC的特性研究; B）肺、骨髓和脑中转移扩散的体内光学成像; C）建立分离的G-MDSC的荧光染料Cy5标记。过继转移后，这些G-MDSC归巢到肺、骨髓和脑中的PMN将通过光学成像可视化。D）建立G-MDSC的PET-（放射性CD11b-单克隆抗体，单纯疱疹-胸苷-激酶-逆转录酶基因）或MRI相容性标记策略（氧化铁颗粒，19F），用于通过同时PET/MRI体内检测它们归巢到PMN。E）在替代的鼠转移模型中通过过继转移的G-MDSC对PMN进行的同时PET/MRI和PMN特异性示踪剂的开发。总之，在本项目中，将研究骨髓和脑中PMN的发育，随后将建立G-MDSC向PMN的迁移动力学，以预测未来转移的位置。拟议的实验将在2015年以后的供资期结束后继续进行。

A significant hallmark of cancer is the appearance of metastasis, frequently representing the final stage of a cancer disease. It is conspicuous, that some tumor types prefere specific organs for metastatic spread. Contrary to obsolete theories, which describe metastasis formation as random event, the metastatic spread is considered nowadays as multi-step process, initiated by the formation of the premetastatic niche (PMN) in the target organ before circulating tumor cells are able to seed. As a consequence of signaling of the primary tumor, an infiltration of different bone marrow derived stem cell types, an increased expression of characteristic adhesion molecules and a remodeling of the extracellular matrix occurs. Hypoxia within the primary tumor induces signaling which results in an increased appearance of PMN, accompanied by an infiltration of granulocytic myeloid derived suppressor cells (G-MDSCs). So far, mainly the development of the PMN in the lung was described in recent publications. In this project the appearance of PMN in the bone marrow and brain will be investigated in a murine breast cancer-metastasismodel (PymT). Specially generated Cherry-red/Luciferase-transgenic-PymT-breastcancer cells with specificity for metastasis in the bone marrow and brain will be used. In these experiments, the treatment of experimental animals with hypoxic conditioned medium derived from bone marrow- or brain-specific breastcancer-cells induces the increased appearance of PMN at the respective sites. This project focuses on the characterization of PMN in the bone marrow and brain, as well as the in vivo imaging of the migration of G-MDSCs to the PMN and can be divided into the following subprojects:During funding period:A) Characterization of G-MDSCs within the PMN of the bone marrow and brain.B) In vivo Optical Imaging of metastatic spread in the lung, bone marrow and brain.C) Establishment of the labeling with the fluorescence dye Cy5 for isolated G-MDSCs. After adoptive transfer, the homing of these G-MDSCs to the PMN in the lung, bone marrow and brain will be visualized by Optical Imaging. After funding period:D) Establishment of PET- (radioactive CD11b-monoclonal antibodies, herpes-simplex-thymidine-kinase-reportergene) or MRI-compatible labeling strategies (iron oxide particles, 19F) for G-MDSCs for the in vivo detection of their homing to the PMN by simultaneous PET/MRI. E) Simultaneous PET/MRI of the PMN by adoptively transferred G-MDSCs in alternative murine metastasis-models and development of PMN-specific tracers. In summary, in this project the development of the PMN in the bone marrow and brain will be investigated and subsequently the migration dynamics of G-MDSCs to PMN will be established to predict the location of the future metastasis. The proposed experiments will sustain after the funding period beyond 2015.