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，以研究完整动物中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，即它们主要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，并且仍然没有被表征。这是因为目前的代谢组学技术不能解析来自混合培养物或组织的各种细胞类型中的每一种的代谢物。因此，目前的方法无法测量肿瘤及其相邻细胞之间的代谢物交换。然而，这些相互作用已被建议定义肿瘤表型。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的事实。纤维二糖由两个葡萄糖分子通过β-键连接而成。我们将对人类成纤维细胞和HeLa细胞系进行基因工程改造，使其能够吸收和消化13 C-纤维二糖（Aim 1）。在13C-纤维二糖中共培养遗传工程化的成纤维细胞与野生型HeLa细胞将使得能够将标记特异性加载到成纤维细胞代谢组中。通过代谢组学对HeLa细胞代谢组进行同位素标记的后续分析将读出代谢物交换（Aim 2）。还将进行相反的实验，其中将遗传工程化的HeLa细胞与野生型成纤维细胞在富含13C-纤维二糖的培养基中共培养。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（Aim 3），将我们的技术扩展到动物肿瘤。在将黑色素瘤细胞移植到斑马鱼中之后，我们将通过用LC/MS和NIMS原位成像追踪同位素标记来跟踪从肿瘤到基质（或反之亦然）的代谢物交换。我们将通过在斑马鱼中使用已建立的细胞特异性启动子来表达血管系统、结缔组织或肌肉中的纤维二糖利用基因，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与其非恶性邻居之间的串扰。该技术的未来应用包括用13C-纤维二糖和基因工程成纤维细胞培养来自患者的活检肿瘤。这将提供一种用于基质饲养的测定，其可以诊断肿瘤表型。其他未来的应用将包括分析小鼠模型中的肿瘤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。这种新陈代谢的有机体观点将对我们理解肿瘤生物学和基础生理学产生深远的影响。

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.