Mechanisms of metastatic outgrowth of spontaneous bone marrow metastases of human prostate cancer cells in vivo

在第一个µBONE资助期内，我们开发了一种小鼠模型，该模型超越了最先进的技术水平，并首次能够对人前列腺癌（PCa）细胞在体内的骨转移级联进行整体建模，从原发性肿瘤的生长开始，以自发形成播散性肿瘤细胞以及向外生长的骨转移结束。我们希望在第二个资助期使用该模型进一步研究PCa骨转移的机制。在我们的模型中，异种移植原发性肿瘤影响骨重建，骨重建的变化（例如由于去势）反过来影响骨转移的形成。Dickkopf相关蛋白1（DKK 1）是一个既调节骨重建又控制转移瘤生长的因子。在我们的初步工作中，shRNA介导的DKK 1的耗竭改变了PCa细胞中EMT/ MET基因的表达，并损害了它们的体外迁移行为。敲除DKK 1受体Krm 1和Krm 2导致NSG小鼠的高骨量，并促进体内自发性PCa骨转移的生长。来自原发性肿瘤、肺转移瘤和骨转移瘤的异种移植亚系在重新异种移植到我们的模型中后表现出不同的克隆异质性和差异转移潜能。我们第二个资助期的目标是进一步探索和转化我们关于DKK 1（共）受体轴在人PCa骨转移瘤转移性生长中的功能作用的研究结果。在完成目前关于DKK 1共受体Krm 1和Krm 2的作用的实验后，我们将分析主要受体Lrp 5在骨转移瘤生长中的作用，并进一步抑制肿瘤细胞本身中的DKK 1。这将使用人源化DKK 1单克隆抗体（hDKN-01）完成，该抗体目前正在PCa患者的1-2期临床试验中进行测试。此外，我们将使用我们组最近在Krm 1/2-KO和/或Lrp 5突变小鼠和/或hDKN-01治疗中建立的新型患者来源的异种移植物（PDX）模型来验证我们的发现。此外，我们将详细分析我们的异种移植原发肿瘤，肺转移和骨转移亚系的功能和分子差异。最后，我们将通过对原发性肿瘤和转移性病变进行激光捕获显微切割，然后进行差异蛋白质组学分析，来利用我们独特的异种移植原发性肿瘤和相应的自发性骨转移资源。通过后两种方法，我们的目标是确定PCa骨转移瘤转移性生长的新功能驱动因素，计划进行临床验证。

In the first µBONE funding period, we developed a mouse model that goes beyond the state of the art and makes it possible for the first time to model the cascade of bone metastasis of human prostate carcinoma (PCa) cells in vivo as a whole, starting with the growth of primary tumors and ending with the spontaneous formation of disseminated tumor cells as well as outgrowing bone metastases. We want to use this model in the second funding period to further investigate the mechanisms of PCa bone metastasis. In our model, the xenograft primary tumors influence bone remodelling and changes in bone remodeling (e.g. due to castration) in turn influence the formation of bone metastases.Dickkopf-related protein 1 (DKK1) is a factor that both regulates bone remodelling and - according to previous work on other tumor entities - controls the outgrowth of metastases. In our preliminary work, shRNA-mediated depletion of DKK1 alters EMT/ MET gene expression in PCa cells and impairs their migration behavior in vitro. Knockout of DKK1 receptors Krm1 and Krm2 leads to high bone mass in NSG mice and promotes the growth of spontaneous PCa bone metastases in vivo. Xenograft sublines from primary tumors, lung metastases and bone metastases show divergent clonal heterogeneity and differential metastatic potential after re-xenografting into our model.Our goals for the second funding period are to further explore and translate our findings on the functional role of the DKK1 (co)-receptor axis in the metastatic outgrowth of human PCa bone metastases. After completion of the current experiments on the role of the DKK1 co-receptors Krm1 and Krm2, we will analyze the role of the main receptor Lrp5 for the outgrowth of bone metastases and furthermore inhibit DKK1 in the tumor cells themselves. This will be done using a humanized DKK1 monoclonal antibody (hDKN-01), which is currently being tested in a phase 1-2 clinical trial in PCa patients. In addition, we will validate our findings using a novel patient-derived xenograft (PDX) model recently established in our group in Krm1/2-KO and/or Lrp5 mutant mice and/or by treatment with hDKN-01. Furthermore, we will analyze in detail the functional and molecular differences of our xenograft primary tumor, lung metastasis and bone metastasis sublines. Finally, we will exploit our unique resource of xenograft primary tumors and corresponding spontaneous bone metastases by performing laser capture microdissections on primary tumor and metastatic lesions, followed by differential proteomics analyses. With the latter two approaches, we aim to identify new functional drivers of metastatic outgrowth of PCa bone metastases, which are planned to be clinically validated.