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：“理解癌症及其环境的异质性”（14-CA-102）。肿瘤间质相互作用被广泛认为是癌症生物学的关键修饰因子，但这种界面的分子成分在很大程度上仍然不确定。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”来调节造血干细胞（hsc）的稳态。白血病“干细胞”（LSCs）也会返回并移植到这个成骨细胞生态位中。不像大多数肿瘤-基质模型，基质成分仍然不明确，成骨细胞是一个高度表征的群体，可以用来定义白血病生态位的潜在的新的调节成分。在这里，我们建议在小生境上使用rna干扰筛选来定义在LSC/成骨细胞和HSC/成骨细胞界面中起功能作用的分子纲要。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）不能在体外分离存活，但当在部分再现体外生态位的成骨细胞单层上共培养时，可以增殖并形成自我更新的“鹅卵石”菌落。我们已经生成了含有“红色”LSCs和“绿色”成骨细胞的共培养物（分别使用表达DS-red和GFP的小鼠）。利用阵列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，并使用我们实验室先前开发的成像软件量化细胞数量和鹅卵石功能。然后将使用功能分析验证这些发夹，并对HSC与LSC效应进行交叉注释。这些实验将提供调节成骨细胞和LSCs/ hsc之间界面的分子途径的概要，从而为在体内研究中测试这些成分创建一个平台。这种“基于小生境”的功能筛选也代表了一种探索肿瘤-基质相互作用的新技术范式。

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.