Measuring signaling pathway dynamics during tissue growth in hydrogels

描述（由申请人提供）：癌症起始和转移进展在体外被广泛建模，应用于这些体外模型的生物材料可以概括体内观察到的表型。我们的长期目标是开发用于定向细胞生长的3D系统，这些研究旨在确定靶向途径以防止异常生长（例如，癌症）或促进功能性组织替代物的发展。我们提出了一种细胞阵列，其报告了在水凝胶中培养的细胞的转录因子（TF）的大规模动态活性，以研究癌基因、材料设计（例如，粘附和降解），和活性TF，以及细胞的最终表型。正常乳腺上皮细胞（MEC）形成能够在允许的环境中产乳的圆形腺泡，而异常癌基因活性（例如，ErbB 2）和特异性细胞外基质环境的存在可产生侵袭前或侵袭性表型。ErbB 2在15-20%的浸润性乳腺癌患者中过表达，在50%的雌激素受体（ER）阴性和12%的ER阳性导管原位癌病例中过表达。所提出的研究集中于驱动从正常表型转变为侵袭前表型（Aim 1），随后从侵袭前表型转变为侵袭表型（Aim 2）的TF。TF是细胞表型的关键调节因子，如其从成纤维细胞产生多能干细胞的能力所证明的，并且它们是信号传导途径的下游靶标，这是许多药物的靶标。TF活性的定量是基于TF报告基因构建体在细胞内的平行递送。 阵列，它与生物发光成像相结合，用于大规模动态定量。TF活性的分析与目前分别定量mRNA或蛋白质丰度的基因组学和蛋白质组学方法不同。这些报告构建体通常在早期时间点应用于少数途径，并且我们的技术允许在水凝胶内培养的细胞的正常和异常结构的整个发育过程中跟踪TF活性。我们采用设计师水凝胶调节粘附和降解，以调查建立依赖表型转换的基质。在目标3中，我们研究了靶向EGFR信号传导的多种药物疗法，如曲妥珠单抗和拉帕替尼。虽然这些化合物正在进行临床研究，但其作用机制和目标患者人群尚不清楚。此外，许多患者对这些化合物产生耐药性，动态分析可以识别代偿途径。对治疗剂的细胞反应部分取决于微环境背景，并且预期这些研究将鉴定获得治疗反应的条件。综上所述，我们假设动态TF活性将i）鉴定与正常和异常组织生长相关的途径特征，ii）提供将水凝胶设计与活性信号传导途径相关的基本设计原则，和iii）鉴定可为临床试验提供信息的药物作用机制。 公共卫生相关性：生物学和疾病进展的体外模型是一个新兴的机会，因为3D培养可以提供更准确的模型，重现体内表型。我们应用一种新的技术进行TF活性的大规模动态分析，以研究由ErbB 2异常活性引起的癌症起始和进展，ErbB 2在15-20%的浸润性乳腺癌患者中过表达，并且由于ErbB 2异常活性引起的侵袭依赖于细胞外基质的性质。这些研究的结果有望确定哪些途径在正常乳腺上皮细胞和乳腺癌细胞中具有活性，以检测从正常表型到侵袭前表型或从侵袭前表型到侵袭表型的转变，这可以确定可能导致控制细胞反应的干预措施的异常途径活性。

DESCRIPTION (provided by applicant): Cancer initiation and metastatic progression are widely modeled in vitro, and biomaterials applied to these in vitro models can recapitulate the phenotypes that are observed in vivo. Our long-term goal is developing 3D systems for directed cell growth, and these studies are aimed at identifying pathways to target to prevent abnormal growth (e.g., cancer) or to promote the development of functional tissue replacements. We propose a cell array that reports on the large-scale dynamic activity of transcription factors (TFs for cells cultured in a hydrogel in order to investigate the relationship between oncogenes, material design (e.g., adhesion and degradation), and the active TFs, and the ultimate phenotype of the cells. Normal mammary epithelial cells (MECs) form round acini capable of milk production in permissive environments, whereas the combination of aberrant oncogene activity (e.g., ErbB2) and the presence of specific extracellular matrix environment can produce pre- invasive or invasive phenotypes. ErbB2 is overexpressed in 15-20% of patients with invasive breast cancer, and in 50% estrogen receptor (ER) negative and 12% of ER positive cases of ductal carcinoma in situ. The proposed studies focus on TFs that drive the transition from a normal to a pre-invasive phenotype (Aim 1), and subsequently from a pre-invasive phenotype to an invasive phenotype (Aim 2). TFs are key regulators of cell phenotype, as evidenced by their ability to generate pluripotent stem cells from fibroblasts, and they are the downstream targets of signaling pathways, which are the target of many pharmaceuticals. The quantification of TF activity is based on the parallel delivery of TF reporter constructs within an array, which is combined with bioluminescence imaging for large scale, dynamic quantification. Analysis of TF activity is distinct from the current genomics and proteomics approaches that quantify mRNA or proteins abundance respectively. These reporter constructs have typically been applied to few pathways at early time points, and our technology allows tracking of TF activity throughout development of normal and abnormal structures for cells cultured within hydrogels. We employ designer hydrogels to regulate adhesion and degradation in order to investigate the established dependence of phenotypic transitions on the matrix. In Aim 3, we investigate multiple drug therapies that target EGFR signaling, such as trastuzumab and lapatinib. While these compounds are being investigated clinically, their mechanism of action and the target patient population are unknown. Furthermore, many patients develop resistance to these compounds, and the dynamic analysis may identify compensatory pathways. The cellular response to therapeutics is dependent, in part, upon the microenvironmental context, and these studies are expected to identify conditions under which a therapeutic response is obtained. Taken together, we hypothesize that the dynamic TF activity will i) identify pathway signatures that correlate with normal and abnormal tissue growth, ii) provide fundamental design principles that relate the hydrogel design to active signaling pathways, and iii) identify mechanisms of drug action that may inform clinical trials. PUBLIC HEALTH RELEVANCE: In vitro models in biology and disease progression are an emerging opportunity, as 3D culture can provide more accurate models that recapitulate in vivo phenotypes. We apply a novel technology for large scale dynamic analysis of TF activity to investigate cancer initiation and progression resulting from aberrant activity of ErbB2, which is overexpressed in 15-20% of patients with invasive breast cancer, and invasion due to aberrant ErbB2 activity is dependent upon the properties of the extracellular matrix. Results from these studies are expected to identify which pathways are active in normal mammary epithelial cells and breast cancer cells to detect the transition from a normal to pre-invasive phenotype, or a pre-invasive to invasive phenotype, which can identify aberrant pathway activity that may lead to interventions that control the cell response.