Metabolic Flux Analysis in Tumor Research

Tumor metabolism is one of the earliest areas of research in tumor biology. Tumor cells have significantly altered their metabolic patterns and metabolic levels compared to normal cells in order to maintain their malignant characteristics, and this change is known as metabolic reprogramming. The reprogrammed tumor metabolism increases the uptake of nutrients such as glucose and glutamine, and extensively utilizes glycolysis and tricarboxylic acid cycle intermediates to synthesize biomolecules. At the same time, the tumor microenvironment interacts with the metabolism of tumor cells. The metabolic reprogramming is characterized differently for different types of tumors. Multiple factors are known to play a role in tumor metabolic reprogramming, including tumor microenvironment (hypoxia, oxidative stress, etc.), activation of oncogenes and their regulated signaling pathways, metabolic enzyme mutations, transcription factors, epigenetic modifications, etc.

The complex mechanisms of tumor metabolism regulation and the numerous influencing factors have limited the advancement of research on tumor metabolism. In recent years, the development of mass spectrometry has provided the possibility of qualitative and quantitative analysis of a wide range of metabolic intermediates and metabolites. Metabolomics uses detection instruments such as mass spectrometry to reflect changes in metabolic pathways by quantifying the abundance of metabolites in biological samples, pointing the way for further mechanistic studies. However, metabolomics reflects a static metabolite abundance. An increase in a particular metabolite alone may be due to either an active synthetic pathway or an inhibition of a depletion pathway. Therefore, for the study of specific metabolic pathways and metabolic networks, metabolomics is often insufficient to explain everything. As a complementary tool to metabolomics, metabolic flow analysis uses stable isotope labeling of specific molecules and traces their metabolic processes in the organism to obtain information on the dynamics of metabolites in metabolic pathways.

Principle of metabolic flow technology

Isotope tracing techniques are one of the important research tools in biology. Early biologists used isotopes to label small molecules and proteins. The important physiological activities such as protein phosphorylation, ubiquitination, cell cycle, and apoptosis could not have been discovered without the contribution of isotope labeling. The combination of isotopic labeling and mass spectrometry detection has made it possible to quantitatively analyze the flow of metabolites through metabolic pathways. Metabolic flow analysis uses stable isotopes to label specific compounds and to deduce the flow and distribution of the compound in the metabolic pathway by analyzing the isotopic labeling pattern of downstream metabolites. By performing metabolic flow analysis on different states of an organism, the degree of activity of a specific metabolic pathway of the organism and the degree of influence of that state on the metabolic pathway can be obtained.

Overview of procedure for high-resolution ¹³C metabolic flux analysis (Long et al., 2019)

Applications of metabolic flow technology in tumor research

Metabolic flow analysis has been widely used in many life science fields such as bioengineering, molecular biology, biochemistry and cell biology. In recent years, the use of metabolic flow analysis to obtain key evidence of metabolic changes has been common in tumor metabolism research.

1. Identify the source of nutrition for tumors

Glucose, lactate and glutamine are important carbon and nitrogen sources for tumor cells. Metabolic flow analysis uses isotopic labeling to establish a link between different metabolites, enabling a more accurate analysis of the nutritional sources of tumors.

2. Reveal tumor-specific metabolism

The extensive use of metabolic flow analysis in tumor metabolism research has not only provided a deeper insight into previously known tumor-specific metabolic pathways, but also identified a variety of new metabolites and metabolic pathways regulating tumorigenesis, progression and invasion.

3. Discover new targets for tumor treatment

A series of key enzymes and genes regulating tumor metabolism have been identified, and IDH is a typical representative of them. The search for new targets is also inseparable from the contribution of metabolic flow analysis.

Poillet-Perez et al. used isotope-labeled arginine to observe the metabolism of arginine in a mouse model of transplanted tumors and demonstrated for the first time that autophagy can regulate tumor growth through arginine metabolism. They also found that arginase 1 (ARG1) could down-regulate arginine levels and thus inhibit tumor growth, and ARG1 became one of the potential targets for tumor therapy.

4. Study tumor resistance and drug sensitivity

Tumor metabolism is closely related to tumor drug resistance and drug sensitivity, which can be studied by metabolic flow analysis techniques. Shukla et al. found that pancreatic cancer cells can upregulate glucose metabolism and pyrimidine anabolism to produce high levels of deoxycytidine triphosphate, the latter of which can reduce gemcitabine concentration through competitive inhibition, resulting in drug resistance.

References:

1. Long, C. P., & Antoniewicz, M. R. (2019). High-resolution 13 C metabolic flux analysis. Nature protocols, 14(10), 2856-2877.
2. Poillet-Perez, L., Xie, X., Zhan, et al. (2018). Autophagy maintains tumour growth through circulating arginine. Nature, 563(7732), 569-573.
3. Shukla, S. K., Purohit, V., et al. (2017). MUC1 and HIF-1alpha signaling crosstalk induces anabolic glucose metabolism to impart gemcitabine resistance to pancreatic cancer. Cancer cell, 32(1), 71-87.