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

本实验室利用三维培养模型研究了基因的表型效应 在乳腺上皮细胞生物学上与乳腺肿瘤发生有关。该模型系统具有 揭示了有趣的细胞生物学行为，在标准单层培养中检测不到， 提供了重要的机制见解的过程和途径，似乎发挥重要作用， 乳腺中肿瘤的发生和发展（例如，管腔的填充，从生长停滞中逃脱， 极性损失等）。在本申请建议的研究中，我们将进一步扩大 这些模型通过检查单个乳腺上皮细胞的命运来研究肿瘤发病机制， 携带与乳腺癌起始或进展相关的遗传改变（例如， 癌基因或肿瘤抑制物的下调）。由于肿瘤是从孤立的克隆变异进化而来的， 单个细胞，而不是在这样的组织内的所有细胞中全局，所提出的实验模型将 更接近地模拟与自发性肿瘤发生和进展相关的自然事件， 使我们能够检查正常细胞和乳腺结构的结构组织的影响， 对致癌损伤引起的表型变化的表达的影响。我们将使用特征良好的 MCF-10A永生化人乳腺上皮细胞模型以及其他体外和体内乳腺上皮细胞模型。 我们正在开发的上皮细胞模型。这些研究将包括系统比较 细胞在增殖与生长停滞结构中受到不同致癌损伤的命运， 单细胞与总细胞群，以及永生化与原代细胞三维 结构.体外模型的发现将在体内使用cDNA的诱导表达或 乳腺内单个细胞中的shRNA。这些研究有望提供重要的信息 （i）携带致癌改变的细胞克隆的命运，（ii）上皮细胞在肿瘤中的作用， 微环境对癌基因损伤结果的影响，（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 组织样结构中的细胞。

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.