Mechanisms associated with organotropic metastasis

摘要 大多数患有实体瘤的癌症患者死于转移性疾病，向器官扩散是一个未充分研究的方面 转移，这迫切需要新的见解。流行的“种子和土壤”概念认为， 远处器官（土壤）中的容许环境对于支持潜伏肿瘤的存活和生长是必要的 种子（Seeds）提出在非转移性器官中诱导允许的污垢以改变转移的路线;然而， 缺乏严格的实验证据和机理分析。纤维化组织改变，包括 炎症，提供转移的线索，以及来自骨髓来源的细胞（BMDC）的信号， 肿瘤分泌体和循环细胞外囊泡。我们的初步数据表明向器官转移 不仅依赖于次级器官中允许的基质重塑和BMDC， 取决于器官特异性血管连接所施加的血管内皮屏障功能的破坏 proteins.我们的研究结果导致了一个中心假设，即“血管异质性在功能上有助于 向器官性转移我们提出研究来解开机制，通过不同的纤维化龛 影响血管床的器官特异性变化，导致向器官转移。初步研究 血管生成素-2（Ang-2）被认为是肺转移的介质。我们的目标是解开 Ang-2依赖于肺而不是肾或肝，肾或肝也在肺中产生高Ang-2水平。 纤维化环境。使用单细胞RNAseq和CyTOF，我们将确定细胞和分子靶点， 转移前环境中的Ang-2。初步研究表明Ang-2可诱导肺血管渗漏 在不影响肾或肝血管的情况下，将肿瘤转移至血管，从而引导转移至肺。外泌体释放 也增加肺中的血管通透性和转移性定植，而不影响 肾脏或肝脏脉管系统。纤维化器官的单细胞RNAseq以及基因工程小鼠 （GEMs），将被用来解开乳腺癌中Ang-2的组织特异性破坏的限速作用 转移使用实验室中产生的新型GEM，我们将追踪转移的谱系特异性产生- 诱导外泌体并通过蛋白质组学分析鉴定向器官性的决定因素。我们的初步 研究表明，血管紧张素-2通过抑制仅在肺中发现的claudin-5来破坏血管屏障 与肾脏和肝脏血管相比，肾脏和肝脏血管具有多个冗余的内皮细胞， claudins整合具有claudin-5和claudin-5报告小鼠的内皮特异性缺失的小鼠模型， 通过器官特异性内皮细胞功能丧失和获得实验的分子分析，我们将 阐明特定的claudins在向器官转移中的功能。将进行分子研究，以确定 Ang-2介导的claudin-5抑制的假定机制和测试是否操纵血管 渗透性可以改变转移的路线，而不管癌症特异性器官的偏好。成功完成 拟议的研究将为转移机制和治疗意义提供新的见解。

ABSTRACT Most cancer patients with solid tumors die of metastatic disease and organotropic spread is an understudied aspect of metastasis, for which new insights are urgently required. The prevailing ‘seed and soil’ concept posits that permissive environment in a distant organ (soil) is necessary to support the survival and growth of lurking tumor cells (seeds). Induction of permissive soil in a non-metastatic organ is proposed to re-route metastasis; however, rigorous experimental evidence and mechanistic analyses are lacking. Fibrotic tissue alterations, including inflammation, provide cues for metastasis, together with the signals from bone marrow-derived cells (BMDCs), the tumor secretome, and circulating extracellular vesicles. Our preliminary data suggest organotropic metastasis is not solely dependent on permissive matrix remodeling and BMDCs in the secondary organs, but is also contingent on the disruption of vascular endothelial barrier function imposed by organ-specific vascular junction proteins. Our findings lead to a central hypothesis that ‘vascular heterogeneity functionally contributes to organotropism of metastasis’. We propose studies to unravel the mechanisms, by which distinct fibrotic niches effect organ-specific changes in the vascular beds, leading to organotropic metastasis. Preliminary studies identified angiopoetin-2 (Ang-2) as a putative mediator of lung metastasis. We aim to unravel the mechanisms of Ang-2 dependent tropism to the lung but not the kidney or liver, which also generate high Ang-2 levels in the fibrotic setting. Using single-cell RNAseq and CyTOF, we will determine the cellular and molecular targets of Ang-2 in the pre-metastatic milieu. Preliminary studies show Ang-2 induces vascular leakage in the lung vasculature without impacting kidney or liver vessels, thus directing metastasis to the lung. Exosomes released by the fibrotic organs also increase vascular permeability and metastatic colonization in the lung, without affecting kidney or liver vasculature. Single-cell RNAseq of fibrotic organs, as well as genetically engineered mice (GEMs), will be used to unravel the rate limiting effect of tissue-specific disruption of Ang-2 in breast cancer metastasis. Using novel GEMs generated in the lab, we will trace lineage-specific production of metastasis- inducing exosomes and identify the determinants of organotropism via proteomic analysis. Our preliminary studies show Ang-2 disrupts vascular barriers through repression of claudin-5 that is found exclusively in the lung vasculature, in contrast with the kidney and liver vessels, which present with multiple, redundant endothelial claudins. Integrating mouse models with endothelial-specific deletion of claudin-5 and claudin-5 reporter mice, and with molecular profiling of organ-specific endothelial cells in loss- and gain-of-function experiments, we will elucidate functions of specific claudins in organotropic metastasis. Molecular studies will be performed to identify putative mechanism of Ang-2 mediated suppression of claudin-5 and test whether manipulation of vascular permeability can re-route metastasis regardless of cancer-specific organ predilection. Successful completion of the proposed studies will provide new insights into mechanism of metastasis and therapeutic implications.