SELECTIVE ISOLATION OF TRANSFORMED BREAST EPITHELIUM

我们在这项建议中的主要目标是利用新奇的文化条件 实现转化细胞的高频选择和增殖 来自(A)乳腺癌的临床标本和(B)人的培养 化学致癌物暴露后的乳腺上皮细胞(HMEC)。 目前使用的协议中的限制严重阻碍了 这方面的进展。我们的新方法将是揭露混合物 正常细胞和假定的转化子(这在两者中都是不可避免的 (A)和(B))到接近模拟 实体瘤的微环境。在这方面，文献提供了 强有力的证据表明，由于肿瘤生长不平衡和 血管形成，氧气和营养的梯度，即使在非常 肿瘤的早期阶段。我们假设肿瘤性或变性 细胞可能存在遗传和/或表观遗传变化，使它们能够 在这些条件下生存并获得渐进的改变 对于肿瘤进展的后续阶段是必不可少的。在文化上，这是 改变对环境的反应可能会提供可能的 变压器组与周围正常组相比具有选择性扩展优势 细胞，与人们认为的在体内发生的方式大致相同。我们将测试 我们在培养系统中的假设，在该系统中，锚定依赖细胞可以 受到自身产生的氧气和葡萄糖梯度的影响。此外，我们 将确定免疫磁学中的相对转化频率 人类管腔亚群和基本亚群的分离种群 乳腺以及HMEC中来自导管和小叶区的 乳房树。我们将用这个新颖的文化体系来问， 在HMEC中实验诱导转化的产率更高 来自非恶性乳腺癌切除组织，包括来自 家族性乳腺癌的高风险。 本申请中提出的创新方法可以提供 用于选择性分离肿瘤细胞的宝贵框架 为了方便对乳腺癌组织的研究 恶性进展的早期阶段。此外，它还将提供 有机会描绘代表In的特定阶段的标记 体外和体内乳腺上皮细胞转化及鉴定 最敏感的目标细胞。最重要的是，这项研究可能会提供一个 快速调查哪些化学或物理试剂单独或 其组合能够增强或降低转换频率， 从而促进了对其病因和发病机制的研究。 其他组织上皮细胞的癌变也是如此。

Our major goal in this proposal is to use novel culture conditions to achieve high-frequency selection and proliferation of transformed cells from (a) clinical specimens of breast carcinoma, and (b) cultures of human mammary epithelial cells (HMEC) after exposure to chemical carcinogens. Limitations in presently used protocols have significantly hampered progress in this area. Our novel approach will be to expose the mixture of normal cells and putative transformants (which is inevitable in both (a) and (b)above) to conditions which closely simulate the microenvironment of solid tumors. In this regard, the literature provides strong suggestive evidence that, due to an imbalance of tumor growth and vascularization, gradients of O2 and nutrients are formed even during very early stages of neoplasia. We hypothesize that neoplastic or transformed cells may harbor genetic and/or epigenetic changes which enable them to survive under these conditions and to acquire progressive alterations essential for subsequent stages of tumor progression. In culture, this altered responsiveness to the environment could provide the putative transformants selective expansion advantage over the surrounding normal cells, much the same way as it is thought to occur in vivo. We will test our hypothesis in a culture system in which anchorage-dependent cells can be subjected to self-created gradients of O2 and glucose. Moreover, we will determine relative transformation frequency in immunomagnetically separated populations of the luminal and basal subsets of the human mammary gland as well as in HMEC from ductal versus lobular areas of the mammary tree. We will use this novel culture system to ask whether the yield of experimentally-induced transformation is higher in HMEC derived from non-malignant mastectomy tissue including specimens from patients at a high risk of familial breast cancer. The innovative approach proposed in this application can provide an invaluable framework for the selective isolation of tumor cells from primary breast carcinoma tissue in order to facilitate studies on the early stages of malignant progression. Additionally, it will provide the opportunity to delineate markers that represent specific stages of in vitro and in vivo mammary epithelial transformation and to identify the most susceptible target cells. Most importantly, this study may provide a rapid means for investigating which chemical or physical agents alone or in combination are able to enhance or reduce transformation frequency, thereby facilitating studies on the etiology and mechanisms of carcinogenesis in epithelial cells of other tissues as well.