Use of Organotypic and Mammary Gland Models to Investigate the Outcomes of Clonal

我们的实验室利用三维培养模型来研究基因的表型效应。 在乳腺上皮细胞生物学上与乳腺肿瘤的发生有关。该模型系统具有 揭示了在标准单层培养中无法检测到的有趣的细胞生物学行为，并具有 提供了对过程和途径的重要机械性见解，这些过程和途径似乎在 乳腺肿瘤的发生和发展(例如，管腔充盈，逃避生长停滞， 极性丧失等)。在这项申请中提出的研究中，我们将进一步扩大 这些模型通过检测单个乳腺上皮细胞的命运来研究肿瘤的发病机制 携带与乳腺癌发生或进展相关的基因改变(例如 癌基因或肿瘤抑制因子的下调)。由于肿瘤是从孤立的克隆改变进化而来的， 单个细胞，而不是在这种组织内的所有细胞中，所提出的实验模型将 更接近于与自发肿瘤启动和进展相关的自然事件，以及 使我们能够检查正常细胞和乳腺结构的建筑组织的影响 关于致癌侮辱引起的表型变化的表达。我们将使用具有良好特征的 MCF-10A永生化人乳腺上皮细胞模型及其他体内外乳腺 我们目前正在开发的上皮细胞模型。这些研究将涉及系统地比较 在增殖和生长受阻结构中，细胞在不同的致癌损伤下的命运 单个细胞与总细胞群体，永生化细胞与原代细胞的三维 结构。体外模型的结果将在体内使用可诱导的cDNA表达或 乳腺内单个细胞中的shRNA。这些研究有望提供重要的信息。 关于(I)携带致癌改变的细胞克隆的命运，(Ii)上皮细胞在 微环境对癌基因损伤结局的影响：(III)逃避抑制的机制 微环境的影响，以及(Iv)赋予肿瘤竞争优势的要求 组织状结构内的细胞。

Our laboratory has utilized a three-dimensional culture model to investigate the phenotypic effects of genes implicated in breast tumorigenesis on the biology of mammary epithelial cells. This model system has revealed interesting cell-biological behaviors that are not detectable in standard monolayer cultures and has provided important mechanistic insights into processes and pathways that appear to play important roles in tumor initiation and progression in the mammary gland (e.g, filling of the lumen, escape from growth arrest, loss of polarity, etc). In the studies proposed in this application, we will further expand the application of these models for studies of tumor pathogenesis by examining the fate of single mammary epithelial cells that carry genetic alterations associated with breast cancer initiation or progression (e.g. expression of oncogenes or downregulation of tumor suppresors). Since tumors evolve from clonal alterations in isolated, individual cells, rather than globally in all cells within such a tissue, the proposed experimental models will more closely mimic the natural events associated with spontaneous tumor initiation and progression, and allow us to examine the influence of normal cells and the architectural organization of mammary structures on expression of phenotypic changes provoked by oncogenic insults. We will use the well-characterized MCF-10A immortalized human mammary epithelial cell model as well as other in vitro and in vivo mammary epithelial cell models that we are currently developing. The studies will involve systematic comparisons of the fate of cells subjected to distinct oncogenic insults in proliferating versus growth-arrested structures, in single cells versus the total cell population, and in immortalized versus primary cell three- dimensional structures. Findings from the in vitro models will be evaluated in vivo using inducible expression of cDNAs or shRNAs in single cells within the mammary gland. These studies promise to provide important information on (i) the fate of cell clones carrying oncogenic alterations, (ii) the role of epithelial cells within the microenvironment on the outcome of oncogene insults, (iii) the mechanisms of escape from suppressive influences of the microenvironment, and (iv) requirements for conferring a competitive advantage to tumor cells within tissue-like structures.