CELL-SPECIFIC ISOTOPE LABELING TO TRACK INTERCELLULAR METABOLITE EXCHANGE IN CANCER

描述(由申请人提供)：我们建议开发一种细胞特异性同位素标记技术来研究完整动物体内的癌症代谢和代谢物交换。我们的技术解决了大多数癌症代谢组学研究的一个主要局限性，即它们大多是在简单的细胞培养系统上进行的。癌细胞与其环境的代谢相互作用在很大程度上被忽视了，仍然没有确定其特征。这是因为目前的代谢组学技术不能分解混合培养物或组织中各种细胞类型的代谢物。因此，目前的方法不能测量肿瘤与其邻近细胞之间的代谢物交换。然而，这些相互作用被认为是定义肿瘤表型的指标。我们利用脊椎动物细胞不吸收或利用碳水化合物纤维二糖这一事实。纤维二糖是由两个葡萄糖分子组成的，它们之间通过一个？键相连。我们将对能够摄取和消化13C-纤维二糖的人成纤维细胞和HeLa细胞系进行基因工程(目标1)。在13C-纤维二糖中将基因工程成纤维细胞与野生型HeLa细胞共培养，将使标记能够特定地加载到成纤维细胞代谢体中。随后通过代谢组学对HeLa细胞代谢体进行同位素标记的分析将读出代谢物交换的读数(目标2)。还将进行反向实验，将基因工程HeLa细胞与野生型成纤维细胞在13C-纤维二糖强化培养液中共同培养。我们将通过构建转基因斑马鱼黑色素瘤细胞系或表达纤维二糖利用基因的转基因斑马鱼将我们的技术扩展到动物肿瘤(目标3)。将黑色素瘤细胞移植到斑马鱼体内后，我们将通过LC/MS和NIMS原位成像追踪同位素标记，跟踪肿瘤与间质之间的代谢物交换(或反之亦然)。我们将通过在斑马鱼中使用已建立的细胞特异性启动子，在血管系统、结缔组织或肌肉中表达纤维二糖利用基因，进一步解决单个基质细胞类型的作用。我们的技术将提供第一个平台来表征癌细胞和它们的非恶性邻居之间的串扰。这项技术的未来应用包括培养来自13C-纤维二糖患者的活组织肿瘤和基因工程成纤维细胞。这将为间质饲养提供一种检测方法，可用于肿瘤表型的诊断。未来的其他应用将包括分析小鼠模型中的肿瘤新陈代谢以及代谢物交换。事实上，这项技术最终可以应用于绘制任何一对动物组织之间交换的代谢物的图谱。这种新陈代谢的生物观将对我们对肿瘤生物学和基本生理学的理解产生深远的影响。

DESCRIPTION (provided by applicant): We propose to develop a cell-specific isotope labeling technology to study cancer metabolism and metabolite exchange in the intact animal. Our technology addresses a major limitation of most metabolomic studies on cancer, namely that they have been mostly performed on simple cell-culture systems. The metabolic interactions of a cancer cell with its environment have been largely ignored and remain uncharacterized. This is because current metabolomic technologies cannot resolve metabolites from each of the various cell types of a mixed culture or tissue. Therefore, current approaches cannot measure metabolite exchange between tumors and their neighboring cells. Yet, these interactions have been suggested to define tumor phenotype. We exploit the fact that vertebrate cells do not take up or utilize the carbohydrate cellobiose. Cellobiose consists of two glucose molecules joined by a ß-linkage. We will genetically engineer human fibroblast and HeLa cell lines that can take up and digest 13C-cellobiose (Aim 1). Co-culturing genetically engineered fibroblasts with wildtype HeLa cells in 13C-cellobiose will enable specific loading of label into the fibroblast metabolome. Subsequent analysis of the HeLa cell metabolome for isotopic label by metabolomics will be readout of metabolite exchange (Aim 2). The converse experiment will also be performed where genetically engineered HeLa cells are co-cultured with wild type fibroblasts in 13C-cellobiose enriched media. We will extend our technology to tumors in animals by constructing transgenic zebrafish melanoma cell lines or transgenic zebrafish expressing the cellobiose-utilization genes (Aim 3). Following transplant of melanoma cells into zebrafish, we will follow metabolite exchange from tumor to stroma (or vice versa) by tracking isotope labels with both LC/MS and NIMS in situ imaging. We will further resolve the role of individual stromal cell types by using established cell-specific promoters in the zebrafish to express cellobiose-utilization genes in vasculature, connective tissue, or muscle. Our technology will provide the first platform to characterize the crosstalk between cancer cells and their nonmalignant neighbors. Future applications of the technology include culturing biopsied tumors from patients with 13C-cellobiose and genetically engineered fibroblasts. This will provide an assay for stromal feeding, which could be diagnostic of tumor phenotype. Additional future applications will include analysis of tumor metabolism in the mouse model as well as metabolite exchange. Indeed, the technology can ultimately be applied to map metabolites exchanged between any pair of animal tissues. This organismal view of metabolism will have profound impacts on our understanding of both tumor biology and basic physiology.