Identification of functional tumor-stromal interactions in the bone marrow

描述(由申请人提供)：本申请涉及广泛的挑战领域“干细胞”和特定领域：“了解癌症及其环境的异质性”(14-CA-102)。肿瘤间质的相互作用被广泛认为是癌症生物学的重要调节因子，但这种界面的分子组成在很大程度上仍未确定。在造血过程中，骨髓中的成骨细胞提供了一个关键的基质“生态位”，调节造血干细胞(HSCs)的稳态。白血病“干细胞”(LSCs)也在这个成骨细胞的缝隙中安家和植入。与大多数肿瘤间质模型不同，成骨细胞是一个高度特征化的群体，可以用来定义白血病生态位潜在的新调节成分。在这里，我们建议使用在壁龛上的RNA干扰屏幕来定义在LSC/成骨细胞和HSC/成骨细胞界面上起作用的分子的概要。我们已经证明，小鼠LSCs(由人类MLL-AF9易位基因的错误表达产生)不能在体外隔离存活，但当共培养在部分概括体外生态位的成骨细胞单层上时，可以增殖并形成自我更新的“鹅卵石”集落。我们已经产生了含有“红色”LSCs和“绿色”成骨细胞的共培养细胞(分别使用DS-red和GFP表达的小鼠)。利用排列的shRNA文库，我们建议敲除“红色”LSCs或“绿色”成骨细胞中的单个基因，并使用我们实验室以前开发的成像软件来量化细胞数量和鹅卵石功能。然后，这些发夹将使用功能分析进行验证，并交叉注释HSC与LSC的效果。这些实验将提供调节成骨细胞和LSCs/HSCs之间接口的分子途径的概要，从而为在体内研究中测试这些成分创造了一个平台。这种基于利基的功能筛选也代表了一种新的技术范式，可以从总体上探索肿瘤与间质的相互作用。 与公共卫生相关：启动肿瘤的干细胞并不孤立地生活，而是居住在由基质细胞创造的被称为“小生境”的高度调控的微环境中；识别和消除这些相互作用是抗癌治疗的一个未被开发的目标，类似于摧毁肿瘤细胞的“家园”。在骨髓中，成骨细胞为白血病细胞创造了一个这样的利基，尽管控制这一界面的分子机制尚不清楚。在这个项目中，我们将开发新的技术来评估数千个基因，以找到那些对白血病干细胞与成骨细胞的功能接触至关重要的基因，希望在未来针对这些相互作用。

DESCRIPTION (provided by applicant): This application addresses broad challenge area "Stem cells" and specific area: "Understanding the Heterogeneity of Cancer and its Environment" (14-CA-102). Tumor stroma interactions are widely recognized to be crucial modifiers of the biology of cancer, but molecular components of this interface have remained largely undefined. In hematopoeisis, osteoblasts in the bone marrow provide a critical stromal "niche" that regulates the homeostasis of hematopoietic stem cells (HSCs). Leukemia "stem cells" (LSCs) also home and engraft in this osteoblastic niche. Unlike most tumor-stroma models, where the stromal component remains poorly defined, osteoblasts are a highly characterized population that can be used to define potentially novel regulatory components of the leukemia niche. Here, we propose to use an RNA-interference screen on the niche to define a compendium of molecules that act functionally in the LSC/osteoblast and the HSC/osteoblast interface. We have shown that murine LSCs (generated by the misexpression of the human MLL-AF9 translocation gene) do not survive in vitro in isolation, but can proliferate and form self-renewing "cobblestone" colonies when cocultured on monolayers of osteoblastic cells that partially recapitulate an ex vivo niche. We have generated co-cultures containing "red" LSCs and "green" osteoblasts (using DS-red and GFP expressing mice respectively). Using an arrayed shRNA library, we propose to knockdown individual genes in either the "red" LSCs or "green" osteoblasts and quantify cell number and cobblestoning function using imaging software previously developed in our labs. These hairpins will then be validated using functional assays and cross-annotated for HSC versus LSC effect. These experiments will provide a compendium of molecular pathways that regulate the interface between osteoblasts and LSCs/HSCs, thus creating a platform for testing these components in in-vivo studies. Such "niche-based" functional screening also represents a novel technological paradigm to explore tumor-stroma interactions in general. PUBLIC HEALTH RELEVANCE: Tumor-initiating stem cells do not live in isolation, but reside in highly regulated microenvironments termed "niches" created by stromal cells; identifying and eradicating these interactions represents an unexploited target for anti-cancer therapy, akin to destroying the "home" of a tumor cell. In the bone marrow, osteoblasts create one such niche for leukemia cells, although the molecular mechanisms that govern this interface are unknown. In this project, we will develop new technologies to evaluate thousands of genes to find those that are essential for the functional engagement of leukemia stem cells with osteoblasts in the hopes of targeting these interactions in the future.