Development of Innovative Human Stromal Invasion Assay

描述（由申请人提供）：本STTR提案的最终目标是开发一种创新的人类肿瘤侵袭试验，作为鉴定新的抗肿瘤治疗方法的筛选工具。需要开发早期肿瘤侵袭模型，即转移的前驱条件，并结合高度敏感的读取，以确定可用于消除人类原发性H&N肿瘤和其他类型癌症侵袭的药物和/或制剂的新靶点。Allen-Hoffmann实验室开发的基于NIKS（tm）的人类肿瘤模型允许监测正常发育的分层鳞状上皮内的肿瘤细胞生长。多光子成像是活体组织无损检测的重要工具，为检测肿瘤底层和/或肿瘤周围基质的早期变化提供了高灵敏度的读数。我们建议扩展我们现有的NIKS(tm)-NTM模型，通过纳入具有基因工程的侵袭性表型的人类肿瘤细胞，并使用多光子成像监测这些细胞的行为。第一阶段将完成以下具体目标：(1)设计并构建MT1-MMP表达载体，验证瞬时转染SCC13y细胞单层培养中MT1-MMP mRNA表达水平升高；(2)验证瞬时转染SCC13y细胞单层培养中蛋白表达水平和生物活性升高；(3)构建稳定的转基因SCC13y细胞克隆，并利用多光子显微镜评估这些稳定克隆的生物活性。新的基质侵袭模型与复杂的基于成像的读数的独特组合将为制药行业提供一种生物学相关的、基于人类细胞的高通量筛选工具，以评估新的细胞抑制剂和/或基因治疗策略。

DESCRIPTION (provided by applicant): The ultimate goal of this STTR proposal is to develop an innovative human tumor invasion assay for use as a screening tool in the identification of new anti-tumor therapeutics. Development of a model of early stage tumor invasion, the precursor condition to metastasis, coupled with a highly sensitive readout, is needed to identify new targets for drugs and/or agents that can be used to abrogate the invasion of primary H&N tumors and other types of carcinoma in humans. The NIKS(tm)-based human tumor model developed in the Allen-Hoffmann laboratory permits monitoring tumor cell growth within a normally developed, stratified squamous epithelium. Multiphoton imaging, an important tool for nondestructive investigation of living tissues, provides a highly sensitive readout for the detection of early changes in stroma underlying and/or surrounding tumors. We propose to expand upon our existing NIKS(tm)-NTM model by incorporating human tumor cells that possess a genetically engineered, invasive phenotype, and monitoring the behavior of these cells using multiphoton imaging. The following specific aims will be accomplished during Phase I: (1) Design and construct MT1-MMP expression vector, and demonstrate elevated MT1-MMP mRNA expression levels in transiently-transfected SCC13y cell monolayer cultures, (2) Demonstrate elevated protein expression levels and bioactivity in transiently-transfected SCC13y cell monolayer cultures, and (3) Develop stable, genetically-modified SCC13y cell clones, and evaluate bioactivity of these stable clones utilizing multiphoton microscopy. The unique combination of the novel stromal invasion model with a sophisticated imaging based readout will provide the pharmaceutical industry with a biologically relevant, human cell based, high throughput screening tool to evaluate novel cytostatic agents, and/or gene therapy strategies.