Characterization and Functional Analysis of Breast Cancer Secreted Exosomes in Ma

描述(由申请者提供)：我们的合作项目扩展了“转移性生态位”的概念，将其作为乳房恶性肿瘤的一个重要决定因素。在过去的十年里，我们加强了对微环境作为肿瘤生长和转移的潜在驱动因素的概念性理解。我们的下一个挑战是剖析导致S乳房及其第二故乡(即它们传播到的第二故乡，以及它们以“休眠”方式等待再次袭击的地方)中易感上皮细胞恶性行为的精确细胞相互作用。基于Lyden博士对转移前生态位的突破性认识和分子解剖，以及Bissell博士对细胞-细胞外基质(ECM)相互作用的开创性分析，确定了正常和恶性行为，以及乳腺肿瘤微环境调节的关键作用，我们设想了原发和继发肿瘤部位内的生态位。这些是由乳腺肿瘤细胞和其间质之间的串扰控制的，这种串扰是由分泌因子介导的，如趋化因子/细胞因子、细胞外基质成分和外切体，它们都包含这两类分子。具体地说，我们假设在原发肿瘤微环境和骨髓微环境中，外切体介导的串扰促进了肿瘤的进展。由于缺乏预测乳腺癌(BC)进展和转移的生物标志物，我们建议从BC患者和健康对照中分离Exosome(研究较少的被膜包围的小囊泡，其中充满可能导致潜在危害的蛋白质和RNA)，以确定Exosome含量是否可以预测患者的分期、转移负担和生存。我们将定义可用于临床诊断并可能提供治疗靶点的乳腺肿瘤来源外体的蛋白质组学特征。我们还将利用三维共培养模型来确定组织结构的恶性进展和伴随的破坏如何影响外切体的释放和潜在恶意内容的整合。这些外切体的功能将通过测量它们激活邻近的“正常”乳腺基质细胞并从这些细胞诱导相互促进肿瘤功能的能力来测试。最后，我们将利用动物模型和3D共培养来测量BC外切体与骨髓细胞的相互作用，以及外切体是否会引起骨髓内转移前壁龛的形成或破坏组织内休眠的壁龛。所有拟议的研究都将通过靶向外切体中的蛋白质含量来确定这样做是否会消除促癌作用，从而保持治疗的重点。我们工作的长期目标是识别包含在外切体中的靶向分子，以阻止肿瘤进展和阻止转移复发。

DESCRIPTION (provided by applicant): Our collaborative project expands on the concept of 'metastatic niche' as an important determinant of malignancy in the breast. Over the past decade, we have enhanced our conceptual understanding of the microenvironment as a potential driver of tumor growth and metastasis. Our next challenge is to dissect the precise cellular interactions that drive malignant behaviors of susceptible epithelial cells in the breast s well as their secondary homes (i.e., those that they disseminate to, and where they sit in a 'dormant' fashion waiting to strike again). Based on Dr. Lyden's groundbreaking recognition and molecular dissection of the pre-metastatic niche and Dr. Bissell's pioneering analysis of cell-extracellular matrix (ECM) interactions specifying normal versus malignant behavior and the crucial role of the microenvironmental regulation of breast tumors, we conceive of niches within primary and secondary tumor sites. These are governed by the crosstalk between breast tumor cells and their stroma that is mediated by secreted factors like chemokines/cytokines, ECM components, and exosomes, which contain both classes of molecules. Specifically, we hypothesize that exosome-mediated crosstalk in the primary tumor microenvironment and within the bone marrow microenvironment facilitates tumor progression. Since biomarkers predictive of breast cancer (BC) progression and metastasis are lacking, we propose to isolate exosomes (poorly studied small vesicles surrounded by membranes and full of proteins and RNA that are released by tumor cells that could cause potential harm) from BC patients and healthy controls to determine whether exosome content are predictive of patient stage, metastatic burden and survival. We will define proteomic signatures of breast tumor-derived exosomes that can be used clinically for diagnosis and may provide therapeutic targets. We will also utilize 3-dimensional co-culture models to determine how malignant progression and concomitant disruption of tissue architecture influences the exosome release and incorporation of potentially malicious content. The function of these exosomes will be tested by measuring their ability to activate neighboring 'normal' breast stromal cells and to elicit reciprocal tumor-promoting functions from these cells. Finally, we will utilize animal models and 3D co-cultures to measure BC exosome interaction with bone marrow cells and whether exosomes elicit formation of pre-metastatic niches within the bone marrow or disrupt dormant niches within the tissue. All the proposed studies will keep a therapeutic focus by targeting protein content within exosomes to determine whether doing so eliminates tumor-promoting effects. The long-term objectives of our work are to identify targetable molecules contained within exosomes to hinder tumor progression and halt metastatic relapse.