Application of RiboTag-seq to Exploration of Tumor Microenvironments

描述（由申请人提供）：这是一项申请，旨在抓住华盛顿大学两种范式转换技术共存的机会，并通过增加第三种技术，将它们结合在一起，以解决生物医学研究中的主要挑战之一-定量描述复杂组织中多种细胞类型表达模式中复杂的发育，生理和病理关系。Morris博士是一名生物化学家/分子生物学家，他开发了“RiboTag mouse”，可以在没有事先细胞分离的情况下分离和分析组织中单个细胞类型的核糖体相关转录组。Shendure博士是一名基因组科学家，他率先开发了第一代测序平台之一，并在开发第二代测序和大规模基因组数据分析（包括RNA-Seq）的新技术方面拥有广泛的专业知识。新的杂交技术- RiboTag-seq -不仅将提供个体细胞类型转录水平的高度定量测量，而且将通过绘制与感兴趣的转录组相关的核糖体的数量和位置，对个体蛋白质的翻译产生可靠的估计。Drs。Morris和Shendure正在与Fred Hutchinson癌症研究中心的Nelson博士合作，Nelson博士是肿瘤-微环境相互作用的专家，他们将应用ribottag -seq分析肿瘤及其邻近细胞环境（即肿瘤微环境）之间的相互作用。在这种情况下，前列腺上皮和间质转录组将在一个明确定义的小鼠模型中分析癌变过程。RiboTag-seq的结果将与并行执行的表达阵列的结果进行比较，并评估两种方法的优缺点。前列腺癌原位分析的结果将导致对两种常用的裸鼠肿瘤异种移植模型和肿瘤细胞与间质成纤维细胞体外共培养模型的评估。该项目将在获得资助后立即部署，并将创造4个新的全职研究职位和一个兼职学生职位。完成后，该项目将为更广泛的肿瘤微环境研究提供基础，更重要的是，允许将RiboTag-seq技术扩展到任何其他感兴趣的复杂组织的检查。

DESCRIPTION (provided by applicant): This is a application to seize the opportunity of the co-existence of two paradigm-shifting technologies at the University of Washington and, by adding a third, bring them together to address one of the major challenges in biomedical research - quantitative descriptions of intricate developmental, physiological and pathological relationships in expression patterns between multiple cell types in complex tissues. Dr. Morris is a biochemist/molecular biologist who has developed the "RiboTag mouse", which enables isolation and analysis of ribosome-associated transcriptomes of individual cell types in a tissue without prior cell separations. Dr. Shendure is a genome scientist who pioneered the development of one of the first next-generation sequencing platforms and has extensive expertise in developing new technologies for second-generation sequencing and analysis of large-scale genomic data, including RNA-Seq. The new hybrid technology - RiboTag-seq - will not only provide highly quantitative measurements of transcript levels in individual cell types, but will yield robust estimates of the translation of individual proteins by mapping the numbers and positions of ribosomes engaged with transcriptomes of interest. Drs. Morris and Shendure are teaming with Dr. Nelson, who is at the Fred Hutchinson Cancer Research Center and an expert in tumor-microenvironment interaction, to apply RiboTag-seq to analyzing reciprocal interactions between a tumor and its adjacent cellular milieu, i.e. the tumor microenvironment. In this instance, prostatic epithelial and stromal transcriptomes will be analyzed during the course of carcinogenesis in a well-defined mouse model. The results from RiboTag-seq will be compared with those from expression arrays performed in parallel and the strengths and weaknesses of the two approaches evaluated. The outcome of this analysis of prostate cancer in situ will lead to evaluation of two commonly used models of neoplasia-tumor xenografts in nude mice and co-culture in vitro of tumor cells and stromal fibroblasts. This project will be deployed immediately upon funding and will create 4 new full-time research positions and a part-time student position. When completed, the project will provide the basis for more expansive studies of tumor microenvironment and, more significantly, allow extension of the RiboTag-seq technology to examination of any other complex tissue of interest. PUBLIC HEALTH RELEVANCE: One of the major challenges in biomedical research is the analysis of intricate developmental, physiological and pathological relationships in gene expression between multiple cell types in complex tissues. This project brings together a biochemist/molecular biologist, a tumor biologist and a genome scientist, along with a paradigm-shifting set of experimental tools that are currently available at the University of Washington, to address the reciprocal interactions between a tumor and its surrounding cellular milieu - the tumor microenvironment. Besides creating access to burning questions in tumor biology, the overriding importance of this project is that it will provide entire through the RiboTag-seq methodology to studies of any other complex tissue of interest.