SELECTIVE ISOLATION OF TRANSFORMED BREAST EPITHELIUM

我们的主要目标是利用新的培养条件， 实现转化细胞的高频率选择和增殖， 来自（a）乳腺癌的临床样本，和（B）人的培养物 乳腺上皮细胞（HMEC）暴露于化学致癌物后。 目前使用的协议中的限制已经显著阻碍了 这方面的进展。 我们的新方法是将混合物 正常细胞和假定的转化体（这是不可避免的， (a)和（B）以上）的条件下， 实体瘤的微环境。在这方面，文献提供了 强有力的证据表明，由于肿瘤生长的不平衡， 血管化，梯度的O2和营养素形成，即使在非常 早期肿瘤我们假设肿瘤或转化 细胞可能具有遗传和/或表观遗传变化， 在这些条件下生存，并获得渐进的变化 对于肿瘤进展的后续阶段至关重要。在文化中，这 对环境的反应性改变可以提供假定的 转化体选择性扩增优于周围正常 细胞，就像它被认为是在体内发生的一样。我们将测试 我们假设在一个培养系统中，贴壁依赖性细胞可以 会受到氧气和葡萄糖的自发梯度的影响。而且我们 将确定免疫磁性中的相对转化频率 人的管腔和基底亚群的分离群体 乳腺以及来自乳腺导管与小叶区域的HMEC 乳腺树我们将用这个新的文化系统来问， 实验诱导转化的产率在HMEC中更高， 来自非恶性乳房切除组织，包括来自患者的样本， 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 选择性分离肿瘤细胞的宝贵框架， 原发性乳腺癌组织，以便于研究 恶性进展的早期阶段此外，它还将提供 有机会描绘代表特定阶段的标记， 体外和体内乳腺上皮转化，并确定 最敏感的靶细胞。最重要的是，这项研究可以提供一个 快速调查哪些化学或物理制剂单独或 组合能够增强或降低转化频率， 从而有助于研究的病因和机制， 在其他组织的上皮细胞中也是致癌的。

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.