Mechanisms of Early Stage Mammary Tumorigenesis

这个项目有几个组成部分。开发新的成像技术，用于检测和表征小鼠中的浸润前和早期浸润性乳腺癌：我们正在开发磁共振（MR）成像、超声（US）成像和活体显微镜（IVM）技术，用于检测小鼠中的早期乳腺癌。开发这些新成像技术的目的有两个：（i）在乳腺癌小鼠模型中进行早期乳腺肿瘤发生的体内表征（见下文下一个项目），因为早期癌症太小，无法通过眼睛或触诊可见，因此成像技术是必要的，以及（ii）使用小鼠模型作为开发可应用于女性的新成像策略的试验台。除了开发新的成像采集技术，我们还在开发多模态鼠标成像工作站，以改善图像分析和数据存储。我们目前正在野生型小鼠、异种移植模型和转基因模型中进行一项评价非对比MR和US成像的研究;在此过程中，我们成功地开发了一种使用琼脂网格进行MR/US/组织学相关性的新策略。在成功完成本试验性研究后，我们将与Paul代顿合作，开始开发用于量化病变血管系统的超声造影增强技术。我们还与Roberto Weigert建立了合作关系，开始小鼠乳腺的IVM。 在新的小鼠乳腺癌模型中研究细胞类型易感性：正常人乳腺可以广泛地表征为由表达细胞角蛋白K18/K8（管腔细胞）和K5/K14（基底细胞和一些上皮细胞）的上皮细胞组成。已知一些中间上皮细胞共表达K5/K18以及其他角蛋白如K19。为了确定细胞类型易感性在乳腺肿瘤发生中的作用，我们开发了小鼠模型，该模型驱动条件T121等位基因的表达，从而功能性地失活pRb途径，已知pRb途径在人乳腺癌中经常改变-在K18、K19和K5调节的控制下针对特定细胞类型（请参见项目1了解这些模型的描述，以及与前列腺特异性Cre系的交叉以驱动前列腺肿瘤发生）。我们目前正在将这些小鼠培育成乳腺特异性Cre系，以驱动细胞类型特异性、乳腺特异性肿瘤发生，并且还将小鼠与p53条件性敲除小鼠杂交，以确定肿瘤表型是否可以随着p53丢失而加速。将解剖具有肿瘤的小鼠并处理组织用于病理学和分子分析。我们还将应用上述开发的成像技术来表征这些不同小鼠模型中早期肿瘤发生和进展的时间尺度。在等待这些小鼠发育的同时，我们正在研究K5、K18和K19细胞在正常小鼠乳腺中的分布。量化病变的物理性质并评估其在进展中的作用：除了研究早期肿瘤的分子和遗传特征外，我们还对确定病变物理性质在浸润前病变进展中的作用感兴趣。通常，大多数成像技术提供了组织的物理特性的直接反映：对于MRI，组织磁弛豫参数T1、T2或质子密度p;对于US，其声学特性。因此，我们目前正在开发的非对比MR和US成像已经提供了物理表征。我们也有兴趣开发先进的技术来量化病变的物理特性-如硬度（来自US弹性成像）和基底膜通透性（来自IVM）。病变的这种物理特征可以用体内成像进行非侵入性评估，因此可以评估这些性质随时间的变化，以确定是否有任何变化可以预测进展。我们会在成功完成上述MR/US试验研究后，展开这项计划。

There are several components to this project. Development of new imaging techniques for detection and characterization of preinvasive and early invasive mammary cancers in mice: We are developing magnetic resonance (MR) imaging, ultrasound (US) imaging, and intravital microscopy (IVM) techniques for the detection of early stage mammary cancers in mice. The purpose of developing these new imaging techniques is two-fold: (i) to perform in vivo characterization of early mammary tumorigenesis in mouse models of breast cancer (see next project below) for which imaging techniques are necessary since early stage cancers are too small to be visible by eye or palpated, and (ii) to use mouse models as a test-bed for developing new imaging strategies that can be applied in women. In addition to developing new imaging acquisition techniques, we are also developing a Mulitmodality Mouse Imaging Workstation to improve image analysis and data storage. We are currently performing a study evaluating noncontrast MR and US imaging in wild-type mice, xenograft models and transgenic models; in doing so, we have successfully developed a new strategy for performing MR/US/histology correlations using an agar grid. Upon successful completion of this pilot study, we will begin to develop US contrast-enhanced techniques for quantifying lesion vasculature in a collaboration with Paul Dayton. We have also established a collaboration with Roberto Weigert to begin IVM of the mouse mamamry glands. Investigate cell-type susceptibility in new mouse models of breast cancer: The normal human breast can be broadly characterized as being comprised of epithelial cells that express cytokeratins K18/K8 (luminal cells) and K5/K14 (basal and some epithelial cells). Some intermediate epithelial cells are known to co-express K5/K18 as well as other keratins such as K19. To determine the role of cell-type susceptibility in mammary tumorigenesis, we have developed mouse models which drive expression of a conditional T121 allele thus functionally inactivating the pRb pathway, known to be frequently altered in human breast cancer--to specific cell types under control of K18, K19 and K5 regulation (please see Project 1 for a description of these models, and crosses with prostate-specific Cre lines to drive prostate tumorigenesis). We are currently breeding these mice to mammary specific Cre lines to drive cell-type-specific, mammary-specific tumorigenesis, and have also crossed the mice to p53 conditional knockout mice to determine if the tumor phenotype can be accelerated with p53 loss. Mice with tumors will be dissected and tissues processed for pathologic and molecular analysis. We will also be applying the imaging techniques developed above to characterize the timescales of early stage tumorigenesis and progression in these different mouse models. While waiting for these mice to develop, we are performing a study of the distribution of K5, K18 and K19 cells in the normal mouse mammary gland. Quantify lesion physical properties and assess role in progression: In addition to studying the molecular and genetic characteristics of early stage tumors, we are also interested in determining the role of lesion physical properties during progression of preinvasive lesions. In general, most imaging techniques provide a direct reflection of the physcial properites of tissues: for MRI, the tissue magnetic relaxation parameters T1, T2 or proton density p; for US, their acoustic properties. Thus, the noncontrast MR and US imaging we are currently developing already provide a physical characterization. We are also interested in developing advanced techniques to quantify physical lesion properties--such as stiffness (from US elastography) and basement membrane permeability (from IVM). Such physical characteristics of lesions can be noninvasively assessed with in vivo imaging, and thus changes in these properties can be assessed over time to determine if any are predictive of progression. We will commence this project upon successful completion of the MR/US pilot study outlined above.