Regulation of metastatic progression by an endothelial-derived factor

我们之前已经确定了一组组织特异性的microRNA，它们通过不同的方式调节转移进展 癌症类型。在乳腺癌和黑色素瘤中，这种转移调节miRNA通路使 癌细胞贪婪地将内皮细胞招募到原发肿瘤部位(PNG等人，自然，2011；Pencheva等人 Al.，Cell，2012)。在这个领域中一个未知的关键是由这样的招募人员提供的信号(S) 影响转移进展的内皮细胞。我们使用了一种创新的不偏不倚的方法， 肿瘤内皮细胞的核糖体在基因上标记有亲和力标签。这使得能够提纯 肿瘤内皮核糖体及其相关转录本，然后进行下一代 测序。这使得我们能够确定Slit2是一种在内皮细胞中被高度诱导的基因 转移细胞。Slit2是神经系统正常建立所必需的轴突引导分子 连通性。我们在同基因模型中的初步证据表明，Slit2在 血管内皮细胞室显著损害肿瘤原发部位的肿瘤转移。我们提出了一个 转移细胞诱导内皮细胞Slit2的模型，Slit2是促进 肿瘤内的癌细胞(低Slit2)向血管系统(高Slit2)迁移，使其能够进入血管内 和转移。这一模型得到了初步临床关联证据的支持，该证据显示 相对于肿瘤间室，内皮细胞室中Slit2的增加与较高的分期有关 转移性复发率高的肿瘤。在本申请中，我们提出了一系列 严格确认这一令人惊讶的模型的补充方法，并进一步从机械上 解剖它。我们将通过内皮或血管内皮细胞的遗传失活来调节癌细胞对SLIT2信号的感知 肿瘤SLIT2在基因启动的癌症进展模型中使用细胞类型特异性基因失活。 我们将使用活体动物多光子显微镜来可视化Slit2驱动的肿瘤跨内皮细胞迁移 和静脉插管。我们的目标是识别感觉Slit2的肿瘤受体，用免疫组织化学方法 方法研究内皮细胞Slit2与人类肿瘤进展和转移的关系 复发，并发现诱导内皮细胞SLIT2的肿瘤衍生信号。最后，我们将应用这些 通过确定临床使用的治疗方法(我们发现诱导SLIT2促进癌症转移)的洞察力。 这项工作有可能对我们对癌症进展机制的理解产生重大影响 建立内皮细胞作为转移的主要协调者。它可能会对人类产生重要影响 由于这一途径控制着高度流行的癌症类型的进展，并与 人类故态复萌。此外，我们使用的细胞类型特异性核糖体图谱方法可能是 更广泛地应用于研究肿瘤微环境中无穷无尽的细胞类型。

We have previously identified sets of tissue-specific microRNAs that regulate metastatic progression by distinct cancer types. In both breast cancer and melanoma, such metastasis regulating miRNA pathways enable cancer cells to avidly recruit endothelial cells into the primary tumor site (Png et al., Nature, 2011; Pencheva et al., Cell, 2012). A key unknown in the field has been the signal(s) that are provided by such recruited endothelial cells that impact metastatic progression. We have used an innovative unbiased approach wherein ribosomes of tumor endothelial cells are genetically marked with an affinity tag. This enables purification of tumor endothelial ribosomes along with their associated transcripts, which then undergo next-generation sequencing. This allowed us to identify Slit2 as a gene significantly induced in endothelial cells by highly metastatic cells. Slit2 is an axon guidance molecule required for the proper establishment of nervous system connectivity. Our preliminary evidence in syngeneic models reveals that genetic inactivation of Slit2 in the endothelial compartment significantly impairs cancer metastasis from the primary tumor site. We propose a model whereby metastatic cells induce Slit2 in endothelial cells, which serves as a signal that promotes migration of cancer cells within the tumor (low Slit2) towards the vasculature (high Slit2), enabling intravasation and metastasis. This model is supported by preliminary clinical association evidence that reveals that increased Slit2 in endothelial compartment relative to the tumoral compartment associates with higher stage tumors that exhibit higher rates of metastatic relapse. In this application, we propose a series of complementary approaches for rigorously confirming this surprising model and further mechanistically dissecting it. We will modulate Slit2 signal sensing by cancer cells through genetic inactivation of endothelial or tumoral Slit2 using cell-type specific genetic inactivation in a genetically initiated model of cancer progression. We will employ live animal multi-photon microscopy to visualize Slit2-driven tumoral trans-endothelial migration and intravasation. We aim to identify the tumoral receptor that senses Slit2, to use immunohistochemical methods to investigate an association between endothelial Slit2 and human cancer progression and metastatic relapse, and to discover the tumor-derived signal that induces endothelial Slit2. Finally, we will apply these insights by determining if a clinically used therapeutic, which we find induces Slit2 promotes cancer metastasis. This work has the potential for major impact on our understanding of mechanisms of cancer progression by establishing endothelial cells as major orchestrators of metastasis. It could have important impact on human disease given that this pathway governs progression of highly prevalent cancer types and associates with human relapse. Moreover, the cell-type specific ribosomal profiling method we have employed could be applied more broadly to study endless cell-types within the tumor microenvironment.