Targeted metabolomics offers new insights into disease states. Knowing that metabolomics strategies are ideal for cancer research, Hu et al. analyzed head and neck cancer cells using a combination of high-performance ion chromatography (IC) separation, high-resolution and accurate-mass (HRAM) measurements, and stable isotope labeling for absolute quantitation. This strategy offered exquisite sensitivity and exceptional reproducibility over a wide, dynamic range.
The team investigated oral cancer stem-like cells (CSCs), non-stem cancer cells (NSCCs), as well as UM1, UM2, UM5, and UM6 head and neck cancer cells. Hu et al. targeted six metabolites, including pyruvate and five TCA intermediates: succinic acid, malic acid, citric acid, fumaric acid and alpha-ketoglutaric acid. To look for new insights into cancer metabolics, the team enriched and isolated CSCs and NSCCs, and then extracted metabolites using liquid nitrogen (LN2) to snap-freeze samples with methanol/water.
Hu and colleagues used stable isotope labeling to spike serially diluted standards into the biological samples for their quantitative analysis. They found that Tracefinder software (Thermo Scientific) was especially helpful for rapidly and accurately quantifying endogenous levels of the six targeted metabolites.
The researchers analyzed the metabolites on a Dionex ICS-5000+ Capillary HPIC System equipped with an anion electrolytic suppressor and coupled with a Q Exactive hybrid quadrupole-Orbtrap mass spectrometer (Thermo Scientific). The team remarked that this strategy was able to provide rapid separation of cellular metabolites within 20 minutes. Another benefit was the exceptional resolving power for polar molecules and isobaric metabolites. To demonstrate this, the team examined the separation of 11 sugar monophosphates and 9 sugar diphosphates present in the UM1 cancer cells. While these sugars can sometimes be difficult to distinguish in IC, the sugars were well-resolved and easily separated using high-resolution IC with Q Exactive mass spectrometry.
After analyzing the head and neck cells, the team found higher levels of TCA metabolites, including malic acid, fumaric acid, citric acid and succinic acid, in UM1 cells compared with UM2 cells. Interestingly, they found UM5 cells expressed significantly higher levels of all six metabolites than the other cell lines, with some metabolites (malic acid, citric acid, fumaric acid and alpha-ketoglutaric acid) at levels two times higher. Additionally, pyruvate levels in UM5 were nearly 20 times higher than those in the other three cell lines. The team posits UM5 cells have a more aggressive metabolic phenotype, particularly the production of pyruvate, which may also be linked to more aggressive metabolic phenotypes found in the highly invasive head and neck cancer cells (UM5 and UM1).
When the team performed a untargeted analysis on the same cells, they found that the results of both targeted and untargeted strategies were in agreement. Specifically, the team found higher levels of pyruvate, citrate, cis-aconitate, isocitrate and 2-oxoglutarate CSCs, whereas succinate, fumarate and malate showed progressively lower levels in CSCs when compared to NSCCs. The team notes that the difference in expressions between CSCs and NSCCs warrants future investigation.
1. Hu, S., et al. (2015) “Targeted metabolomic analysis of head and neck cancer cells using high performance ion chromatography coupled with a Q Exactive HF mass spectrometer,” Analytical Chemistry 87(12) (pp. 6371–9), doi: 10.1021/acs.analchem.5b01350.