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Molecular & Cellular Analysis Technologies
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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 á-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.