Development of Innovative Human Stromal Invasion Assay

创新型人类基质侵袭试验的开发

• 批准号: 6993325
• 负责人: B LYNN ALLEN-HOFFMANN
• 金额: $ 10万
• 依托单位: STRATATECH CORPORATION
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-29 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6993325
• 关键词: antineoplastics; bioassay; cell biology; cell line; clone cells; cytogenetics; drug discovery /isolation; drug screening /evaluation; fluorescence microscopy; gene therapy; head /neck neoplasm; high throughput technology; method development; molecular /cellular imaging; neoplasm /cancer invasiveness; nucleic acid sequence; pharmacokinetics; squamous cell carcinoma; stromal cells; tissue /cell culture; transfection /expression vector

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.

描述（由申请人提供）：该 STTR 提案的最终目标是开发一种创新的人类肿瘤侵袭测定，用作鉴定新抗肿瘤疗法的筛选工具。需要开发早期肿瘤侵袭模型（转移的先兆条件），并结合高度灵敏的读数，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/或药剂的新靶点。 Allen-Hoffmann 实验室开发的基于 NIKS(tm) 的人类肿瘤模型可以监测正常发育的复层鳞状上皮内肿瘤细胞的生长。多光子成像是活体组织无损研究的重要工具，为检测肿瘤下方和/或周围基质的早期变化提供了高度灵敏的读数。我们建议通过纳入具有基因工程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，来扩展我们现有的 NIKS(tm)-NTM 模型。第一阶段将实现以下具体目标：(1) 设计和构建 MT1-MMP 表达载体，并证明瞬时转染的 SCC13y 细胞单层培养物中 MT1-MMP mRNA 表达水平升高，(2) 证明瞬时转染的 SCC13y 细胞单层培养物中蛋白质表达水平和生物活性升高，以及 (3) 开发稳定的基因修饰载体 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。新型基质侵袭模型与基于复杂成像的读数的独特组合将为制药行业提供生物相关的、基于人类细胞的高通量筛选工具，以评估新型细胞抑制剂和/或基因治疗策略。