Development of Experimental and Theoretical Methodology for Determination of Characteristics of central Metabolism Regulation in Cancer Cells

Tracing cellular metabolites with 13C isotope is the state-of-the-art experimental technique to gather information about the distribution of fluxes in biochemical networks in living cells. It is widely applied to study cancer cell metabolism. The isotopic isomer distribution measured in the products of cells incubated with 13C labeled substrates can be viewed as a fingerprint characterizing metabolic fluxes in the studied cells. Such fingerprint can be deciphered using software tools that simulate metabolic fluxes calculating the corresponding isotopomer distribution. However, there are a number of problems arising in conjunction with the identification of metabolic fluxes. One of such problems is that the system of fluxes often is under determined, i.e. the measured set of isotopomers is consistent with various sets of fluxes. Thus, fluxes cannot be determined unambiguously.

One can see two ways of solving this problem, either to measure isotopomer distribution in a larger number of metabolites, or to chose the conditions of cell incubation so that maximally restrict the number of combinations of fluxes consistent with the measurements. Thus, prior to the main project we started the development of methodology for unambiguous flux profile determination exploring the latter way.

When cells are incubated in complete media containing, except the 13C-labeled substrate, various amino acids, and other sources of carbon, multiple possible combinations of various substrates complicates unambiguous flux profile determination. Therefore, the possibility of metabolite labeling incubating cells in minimal media containing, in the best case, only one, labeled, source of carbon is explored. Various substrates can activate different metabolic pathways in the studied cells; therefore the greater variability in the substrates used separately, the more complete picture of cell metabolism can be obtained.

The change of incubation conditions from the complete media, where cells grow, to minimal media can induce different changes in gene expression. Therefore, if we are interested in metabolic response of the cell growing in the same complete media, the period of cell incubation with labeled substrates in minimal media must be short sufficiently to outstrip the manifestation of the changes in gene and protein expression.

Taking into account the above-mentioned ideas for the methodology development, the possibility of cell incubation in the media containing as only source of carbon either glucose, or glutamate or lactate was studied in the HT29 cell line chosen as the best object for the methodological development. The cell survival, substrate consumption, and product accumulation was measured in 3, 6, 24 hours after the starting incubation in minimal media. The results confirm that in 3 hours of incubation the cells survive and consume substrate in sufficient amount to be measured and to study labeled product distribution.

The next step necessary to accomplish this methodology will consist of cell incubation with the labeled substrates in various combinations, measuring isotopomer distributions in products using gas chromatography/mass spectrometry, and the analysis using our software Isodyn to evaluate the distribution of metabolic fluxes in various conditions of incubation.

The knowledge of flux distributions under various conditions will allow us to evaluate the characteristics of regulation of cell metabolism.