Reactivation of breast cancer micrometastases by senescent bone marrow stroma

描述（由申请人提供）：超过三分之一的I-III期乳腺癌患者在诊断时存在骨髓微转移，这是复发的来源。大多数复发发生在绝经后妇女。休眠和复发的机制尚不清楚，但数据表明与骨髓基质密切相关。我们假设基质细胞由于衰老和/或绝经后雌激素剥夺而经历衰老，并开始分泌炎症细胞因子，刺激休眠的癌细胞重新唤醒。我们研究的广泛和长期目标是确定控制骨髓中乳腺癌细胞休眠状态建立的机制，并确定导致其重新唤醒和疾病复发的因素和机制。我们建议在体外和体内小鼠模型中确定骨髓基质在缺乏雌激素或细胞毒素处理时是否会发生衰老。我们的具体目标是：1。为了确定体外雌激素剥夺是否可以诱导不能支持乳腺癌休眠的骨髓基质培养的衰老表型，在体外模型和2。确定体内雌激素剥夺是否在骨髓基质中诱导衰老表型，使其无法在体外和体内支持乳腺癌休眠。我们将通过使间质单层受到氧化和缺氧应激以及雌激素剥夺来建立和表征分泌性衰老的表型，并测量TGF¿，Cox-2, IL-6， IL-8和SA-¿Gal这些已知与衰老相关的标志物的表达和激活。我们将在体外克隆共培养模型和左心室注射骨髓转移模型中确定体外和体内雌激素剥夺以及体外细胞毒性是否可以通过这些分子标记和乳腺癌休眠支持的丧失来诱导衰老。实验还将确定雌激素剥夺是否使基质更容易受到化学损伤，以及使用Cox-2抑制剂或雌激素是否可以逆转这些影响。这些研究将建立一种思考休眠作为衰老微环境功能的方法，并寻求逆转雌激素剥夺引起的炎症以维持休眠。公共卫生相关性：拟议的研究将在体外和体内研究雌激素剥夺对小鼠骨髓基质衰老的诱导，在体外模型中表现为炎症细胞因子的分泌和支持乳腺癌细胞休眠能力的丧失，以及在骨髓微环境中支持异种移植的人乳腺癌细胞休眠能力的丧失。实验将确定用雌激素或抗炎剂治疗是否能恢复衰老基质支持休眠的能力。

DESCRIPTION (provided by applicant): More than a third of stage I-III breast cancer patients have bone marrow micrometastases at the time of diagnosis providing a source of recurrence. Most recurrences occur in post-menopausal women. Mechanisms of dormancy and recurrence are not well understood, but data suggest a dependence on a close association with bone marrow stroma. We hypothesize that stromal cells undergo senescence due to aging and/or post-menopausal estrogen deprivation and begin to secrete inflammatory cytokines that can stimulate dormant cancer cells to re-awaken. The broad, long-term goals of our investigations are to define mechanisms that govern the establishment of the dormant state in breast cancer cells in the bone marrow and to determine factors and mechanisms responsible for their re-awakening and recurrence of disease. We propose to determine if bone marrow stroma can undergo senescence when deprived of estrogen or treated with cytotoxins in vitro and in vivo in a murine model. Our specific aims are: 1. to determine if in vitro estrogen deprivation can induce a senescent phenotype in bone marrow stromal cultures incapable of supporting breast cancer dormancy in an in vitro model and 2. to determine if in vivo estrogen deprivation induces a senescent phenotype in bone marrow stroma rendering it incapable of supporting breast cancer dormancy in vitro and in vivo. We will establish and characterize the phenotype of secretory senescence by subjecting stromal monolayers to oxidative and hypoxic stress and estrogen deprivation and measure the expression and activation of TGF¿, Cox-2, IL-6, IL-8 and SA-¿Gal, known markers associated with senescence. We will determine if estrogen deprivation in vitro and in vivo and cytotoxicity in vitro can induce senescence measured by these molecular markers and by the loss of support of breast cancer dormancy in an in vitro clonogenic co-culture model and in a left ventricle injection bone marrow metastasis model. Experiments will also determine whether estrogen-deprivation renders stroma more susceptible to chemical injury and whether administration of Cox-2 inhibitors or estrogen can reverse these effects. These studies will establish a way of thinking about dormancy as a function of the senescent microenvironment and seek to reverse estrogen-deprivation-induced inflammation to maintain it. PUBLIC HEALTH RELEVANCE: The proposed study will investigate the induction of senescence in mouse bone marrow stroma by estrogen deprivation in vitro and in vivo as manifested by the secretion of inflammatory cytokines and loss of the capacity to support dormancy of breast cancer cells in an in vitro model and the loss of the capacity to support the dormancy of xenografted human breast cancer cells in the bone marrow microenvironment. Experiments will determine if treatment with estrogen or anti-inflammatory agents can restore the capacity of senescent stroma to support dormancy.