Application of the Hydrophilic Interaction Liquid Chromatography (HILIC-MS) Novel Protocol to Study the Metabolic Heterogeneity of Glioblastoma Cells

Abstract

Glioblastoma is a highly malignant brain tumor consisting of a heterogeneous cellular population. The transformed metabolism of glioblastoma cells supports their growth and division on the background of their milieu. One might hypothesize that the transformed metabolism of a primary glioblastoma could be well adapted to limitations in the variety and number of substrates imported into the brain parenchyma and present it their microenvironment. Additionally, the phenotypic heterogeneity of cancer cells could promote the variations among their metabolic capabilities regarding the utilization of available substrates and release of metabolic intermediates. With the aim to identify the putative metabolic footprint of different types of glioblastoma cells, we exploited the possibility for separation of polar and ionic molecules present in culture media or cell lysates by hydrophilic interaction liquid chromatography (HILIC). The mass spectrometry (MS) was then used to identify and quantify the eluted compounds. The introduced method allows the detection and quantification of more than 150 polar and ionic metabolites in a single run, which may be present either in culture media or cell lysates and provide data for polaromic studies within metabolomics. The method was applied to analyze the culture media and cell lysates derived from two types of glioblastoma cells, T98G and U118. The analysis revealed that even both types of glioblastoma cells share several common metabolic aspects, and they also exhibit differences in their metabolic capability. This finding agrees with the hypothesis about metabolic heterogeneity of glioblastoma cells. Furthermore, the combination of both analytical methods, HILIC-MS, provides a valuable tool for metabolomic studies based on the simultaneous identification and quantification of a wide range of polar and ionic metabolites-polaromics.

Keywords: HILIC; LC-MS; amino acid; glioblastoma; metabolic heterogeneity; metabolomics.

Figure 1

Representative chromatogram of of α-ketoisocaproic acid (1), α-ketoisovaleric acid (2), isoleucine (3), leucine (4) and valine (5), on four different columns—Sequant^® ZIC^® cHILIC (A), Sequant^® ZIC^® pHILIC (B), Raptor polar X (C) and YMC-triart Diol-HILIC (D). Compounds were detected according to their m/z values (m/z = 129.0582, grey line; m/z = 115.0428, yellow line; m/z = 132.1019, blue line; m/z = 118.0863, orange line) and retention times. Mass concentration of all compounds was 1 μg/mL.

Figure 2

Representative chromatogram of isoleucine (1, blue), leucine (2, blue) and isotopically labeled ¹³C₆,¹⁵N-leucine (3) separation on Sequant^® ZIC^® cHILIC column. The m/z values (m/z = 132.1019, blue line; m/z = 139.1191, orange line) were used for identification after elution.

Figure 3

Metabolic changes in culture medium of two glioblastoma cell lines (T98G and U118) incubated for 24 h. (A) Heat map, where data are expressed as average of the fold change of metabolites in medium without cells and metabolites in medium after incubation. Metabolites depicted with a blue color are taken up from the medium and metabolites depicted with a red color are released into the medium. (B) Increase in 5-methylthioadenosine and 4-methyl-2-oxopentanoate/3-methyl-2-oxopentanoate in culture medium after incubation. These are depicted here in separate graphs because it was not possible to calculate the fold change, since the two were not detected in the medium without cells. Blue color represents the T98G cell line, and red represents the U118 cell line. Numbers of replicates for each sample is three (n = 3). Error bars are expressed as SEM.

Figure 4

Changes of metabolites in culture medium of T98G (blue) and U118 (red) cell lines. Bar graphs of metabolites indicating specific uptake or release after 24 h of incubation. Numbers of replicates for each sample is three (n = 3). Error bars are expressed as SEM. One asterisk means p < 0.05, two asterisks p < 0.01 and four asterisks p < 0.0001.

Figure 5

Principal component analysis (PCA) score plot of three replicates of two different groups, from all metabolites detected in collected culture media (A) or cell lysates (B) of both types of glioblastoma cells, T98G (red) or U118 (green).

Figure 6

Intracellular content of metabolites in two types of glioblastoma cells, T98G and U118. (A) Heatmap represents the estimated average amount of substance in micromole per mg of lysate proteins. (B) The relative levels of glucose and carnitine are presented on separate graphs since their content, in lysates, was quantified only relatively as the signal of the compound to the peak area of the internal standard. Two cell lines were compared: T98G (blue) and U118 (red). Relative quantification is expressed as a ratio of the peak area of analyte to the peak area of the internal standard. Numbers of replicates for each sample is three (n = 3). Error bars are expressed as SEM.

Figure 7

Estimation of the intracellular levels of metabolites in cultured T98G (blue column) and U118 (red) glioblastoma cells by LC-MS. The metabolites were quantified in lysates derived from culture cells. Bar graphs representing the molar amount of substance standardized per one mg of lysates proteins. Numbers of replicates for each sample is three (n = 3). Error bars are expressed as SEM. One asterisk means p < 0.05, two asterisks p < 0.01.