Isolation and Characterization of ER+ Breast Cancer Cells with High Bone Metastat

说明(申请人提供)：雌激素受体阳性(ER+)腔内乳腺癌占所有乳腺癌病例的75%，是乳腺癌中最有可能转移到骨的亚型，形成无法治愈的溶解病变，在大多数情况下也是ER+。与之形成鲜明对比的是，研究乳腺癌骨转移(B-MET)发病机制和治疗的主流临床前模型使用了人类乳腺癌细胞的ER基础亚型。在缺乏标准的ER+腔B-MET模型的情况下，关于这些肿瘤细胞在骨微环境中的生物学的基本问题是无法质疑的，包括肿瘤细胞雌激素信号在促进骨微环境中的转移进展中的可能作用。这项建议的目的是利用体内选择和普遍研究的ER+管腔人乳腺癌细胞株来建立ER+裂解B-MET的强大临床前模型，然后使用功能基因组学来询问该模型，以检查在这种情况下骨转移的发病机制，并将这些发现与以前在ER-基底细胞模型中获得的结果进行比较和对比。在特定目标1中，通过体内选择，将具有形成裂解B-MET的高转移潜能的不同的ER+细胞亚群通过心内(IC)接种异种人ER+腔癌细胞(MCF-7、ZR-75-1或T47D)到裸鼠体内。这些研究将利用并建立在我们实验室已经使用这种方法分离的独特的嗜骨ER+MCF-7来源细胞系的基础上。在特定的目标2，即功能基因组研究中，将通过比较特定细胞(如目标1所述的嗜骨细胞)和其衍生的“低转移”细胞中的基因表达，确定其结构性或雌激素刺激的表达与骨转移“生态位”中的肿瘤进展和骨溶解相关的基因。体内试验确定雌激素依赖性和这些促骨细胞肿瘤进展增强的部位特异性将进一步有助于解释与溶骨ER+相关基因的功能意义 鲁米那B-MET表型。实现这些目标的成功可能是因为：1)我们正在总结用于确定ER-基底细胞乳腺癌B-MET发病机制的实验方法，以及2)我们已经分离出了嗜骨性ER+MCF-7细胞，并在初步实验中在IC接种ER+T47D细胞的裸鼠中鉴定出了溶骨性B-MET。这些研究的意义在于它们的新颖性，因为还没有开发出用于功能基因组研究中询问的ER+溶血B-MET的管腔模型，以及它们与乳腺癌的临床相关性，因为这些研究解决了一种常见的临床情况，没有治愈的方法。我们的最终目标是通过改进预防和治疗ER+管腔性乳腺癌B-MET的策略，提高被诊断为乳腺癌的女性的存活率，方法是目前和未来使用在这里建议的R03研究中获得的新工具和信息。

DESCRIPTION (provided by applicant): Estrogen receptor-positive (ER+) luminal breast cancers, which account for 75% of all breast cancer cases, are the subtype of breast cancer most likely to metastasize to bone, forming incurable lytic lesions that are, in a majority of case, also ER+. In contradistinction, prevailing pre-clinical models investigating the pathogenesis and treatment of breast cancer bone metastases (B-MET) employ ER- basal subtypes of human breast cancer cells. In the absence of standard models of ER+ luminal B-MET, basic questions regarding the biology of these tumor cells within the bone microenvironment cannot be queried, including a possible role for tumor cell estrogen signaling in promoting metastasis progression within the bone microenvironment. The objective of this proposal is to utilize in vivo selection and commonly studied ER+ luminal human breast cancer cell lines to establish robust pre-clinical models of ER+ lytic B-MET which will then be queried, using functional genomics, to examine the pathogenesis of bone metastasis in this setting, comparing and contrasting these findings with those previously obtained in ER- basal cell models. In Specific Aim 1, distinct subpopulations of ER+ cells with a high metastatic potential for formation of lytic B-MET will be isolated by in vivo selection following intracardiac (IC) inoculation of heterogeneous populations of "low-metastatic" human ER+ luminal breast cancer cells (MCF-7, ZR-75-1, or T47D) into nude mice. These studies will take advantage of, and build upon, unique osteotropic ER+ MCF-7-derived cell lines already isolated by our laboratory using this approach. In Specific Aim 2, functional genomic studies, comparing gene expression in cells specifically selected for their bone tropism during the final stage of metastatic progression (osteotropic cells, as described in aim 1) vs. "low- metastatic" cells from which they are derived, will be undertaken to identify genes whose constitutive or estrogen-stimulated expression is associated with tumor progression and osteolysis in the bone metastatic "niche". In vivo testing to determine estrogen-dependency and site-specificity of enhanced tumor progression for these osteotropic cells will further aid in interpreting the functional significance of genes associated with the osteolytic ER+ luminal B-MET phenotype. Success in achieving these aims is likely given that: 1) we are recapitulating the experimental approach used to determine the pathogenesis of ER- basal cell breast cancer B-MET, and 2 ) we have already isolated osteotropic ER+ MCF-7 cells and identified osteolytic B-MET in nude mice inoculated IC with ER+ T47D cells in preliminary experiments. The significance of these studies lies in their novelty, as ER+ luminal models of lytic B-MET have not been developed for interrogation in functional genomic studies, and their clinical relevance to breast cancer, as these studies address a common clinical situation for which there is no cure. Our ultimate goal is to increase survivorship in women diagnosed with breast cancer by improving strategies to prevent and treat ER+ luminal breast cancer B-MET through the current and future use of the novel tools and information acquired in the R03 studies proposed here.