In cancer tumors, lactate accumulation was initially attributed to high glucose consumption associated with the Warburg Effect. Now it is evident that lactate can also serve as an energy source in cancer cell metabolism. Additionally, lactate has been shown to promote metastasis, generate gene expression patterns in cancer cells consistent with “cancer stem cell” phenotypes, and result in treatment resistant tumors. Therefore, the goal of this work was to quantify the impact of lactate on metabolism in three breast cell lines (one normal and two breast cancer cell lines—MCF 10A, MCF7, and MDA-MB-231), in order to better understand the role lactate may have in different disease cell types. Parallel labeling metabolic flux analysis ( 13 C-MFA) was used to quantify the intracellular fluxes under normal and high extracellular lactate culture conditions. Additionally, high extracellular lactate cultures were labelled in parallel with [U- 13 C] lactate, which provided qualitative information regarding the lactate uptake and metabolism. The 13 C-MFA model, which incorporated the measured extracellular fluxes and the parallel labeling mass isotopomer distributions (MIDs) for five glycolysis, four tricarboxylic acid cycle (TCA), and three intracellular amino acid metabolites, predicted lower glycolysis fluxes in the high lactate cultures. All three cell lines experienced reductive carboxylation of glutamine to citrate in the TCA cycle as a result of high extracellular lactate. Reductive carboxylation previously has been observed under hypoxia and other mitochondrial stresses, whereas these cultures were grown aerobically. In addition, this is the first study to investigate the intracellular metabolic responses of different stages of breast cancer progression to high lactate exposure. These results provide insight into the role lactate accumulation has on metabolic reaction distributions in the different disease cell types while the cells are still proliferating in lactate concentrations that do not significantly decrease exponential growth rates.

DNA microarray analysis of gene expression has become a valuable tool for bioprocessing research aimed at improving therapeutic protein yields. The highly parallel nature of DNA microarray technology allows researchers to assess hundreds of gene simultaneously, essentially enabling genome-wide snapshots. The quality and amount of therapeutic proteins produced by cultured mammalian cells rely heavily on the culture environment. In order to implement beneficial changes to the culture environment, a better understanding of the relationship between the product quality and culture environment must be developed. By analyzing gene expression levels under various environmental conditions, light can be shed on the underlying mechanisms. This paper describes a method for evaluating gene expression changes for cultured NS0 cells, a mouse-derived myeloma cell line, under culture environment conditions, such as ammonia buildup, known to affect product quality. These procedures can be easily adapted to other environmental conditions and any mammalian cell lines cultured in suspension, so long as a sufficient number of gene sequences are publicly available.

NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, the of these genes was analysed in response to various culture conditions – elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analysed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium.

Stem cells are needed for an increasing number of scientific applications, including both fundamental research and clinical disease treatment. To meet this rising demand, improved expansion methods to generate high quantities of high quality stem cells must be developed. Unfortunately, the bicarbonate buffering system – which relies upon an elevated CO2 environment – typically used to maintain pH in stem cell cultures introduces several unnecessary limitations in bioreactor systems. In addition to artificially high dissolved CO2 levels negatively affecting cell growth, but more importantly, the need to sparge CO2 into the system complicates the ability to control culture parameters. This control is especially important for stem cells, whose behavior and phenotype is highly sensitive to changes in culture conditions such as dissolved oxygen and pH. As a first step, this study developed a buffer to support expansion of mesenchymal stem cells (MSC) under an atmospheric CO2 environment in static cultures. MSC expanded under atmospheric CO2 with this buffer achieved equivalent growth rates without adaptation compared to those grown in standard conditions and also maintained a stem cell phenotype, self-renewal properties, and the ability to differentiate into multiple lineages after expansion.

SWH) ¶ These authors contributed equally to this work High extracellular lactate increases reductive carboxylation 2 1 Abstract 2 In cancer tumors, lactate accumulation was initially attributed to high glucose consumption 3 associated with the Warburg Effect. Now it is evident that lactate can also serve as an energy source in 4 cancer cell metabolism. Additionally, lactate has been shown to promote metastasis, generate gene 5 expression patterns in cancer cells consistent with "cancer stem cell" phenotypes, and result in treatment 6 resistant tumors. Therefore, the goal of this work was to quantify the impact of lactate on metabolism in 7 three breast cell lines (one normal and two breast cancer cell lines -MCF 10A, MCF7, and MDA-MB-8 231), in order to better understand the role lactate may have in different disease cell types. Parallel labeling 9 metabolic flux analysis ( 13 C-MFA) was used to quantify the intracellular fluxes under normal and high 10 extracellular lactate culture conditions. Additionally, high extracellular lactate cultures were labelled in 11 parallel with [U-13 C] lactate, which provided qualitative information regarding the lactate uptake and 12 metabolism. The 13 C-MFA model, which incorporated the measured extracellular fluxes and the parallel 13 labeling mass isotopomer distributions (MIDs) for five glycolysis, four tricarboxylic acid cycle (TCA), and 14 three intracellular amino acid metabolites, predicted lower glycolysis fluxes in the high lactate cultures. All 15 three cell lines experienced increased reductive carboxylation of glutamine to citrate in the TCA cycle as a 16 result of high extracellular lactate. Increased reductive carboxylation previously has been observed under 17 hypoxia and other mitochondrial stresses, whereas these cultures were grown aerobically. In addition, this 18 is the first study to investigate the intracellular metabolic responses of different stages of breast cancer 19 progression to high lactate exposure. These results provide insight into the role lactate accumulation has on 20 metabolic reaction distributions in the different disease cell types while the cells are still proliferating in 21 lactate concentrations that do not significantly decrease exponential growth rates. 23 IntroductionHigh extracellular lactate increases reductive carboxylation 3 24 Since the 1920s, many types of cancers have been shown to rely heavily on glycolysis and lactate 25 fermentation to produce energy rather than the more energy efficient complete oxidation of glucose in the 26 mitochondria, even in the presence of sufficient oxygen. This metabolic state is called the Warburg Effect 27 [1][2][3][4]. In addition, lactate can be utilized by cancer cells in the presence of glucose, a process known as the 28 Reverse Warburg Effect [5][6][7][8][9]. Not only does this capability to use lactate provide cancer cells a metabolic 29 advantage in vivo, it seems to favor cancer progression. For example, when lactate was injected into mice 30 with xenografts of the human breast cancer ...