Use of MRI, metabolomic, and genomic biomarkers to identify mechanisms of chemoresistance in glioma

Abstract

Gliomas are the most common form of central nervous system tumor. The most prevalent form, glioblastoma multiforme, is also the most deadly with mean survival times that are less than 15 months. Therapies are severely limited by the ability of these tumors to develop resistance to both radiation and chemotherapy. Thus, new tools are needed to identify and monitor chemoresistance before and after the initiation of therapy and to maximize the initial treatment plan by identifying patterns of chemoresistance prior to the start of therapy. Here we show how magnetic resonance imaging, particularly sodium imaging, metabolomics, and genomics have all emerged as potential approaches toward the identification of biomarkers of chemoresistance. This work also illustrates how use of these tools together represents a particularly promising approach to understanding mechanisms of chemoresistance and the development individualized treatment strategies for patients.

Keywords: Sodium MRI; diffusion; genes; glycolysis; resistance; warburg effect.

Figure 1

Development of BCNU-resistant 9L glioma cells. BCNU sensitive 9L glioma cells (9L-S) were grown in the presence of increasing concentrations of BCNU resulting in a 9L subculture that was BCNU-resistant (9L-R). Cell viability of 9L-S and 9L-R cells was quantified after exposure of both cell types to increasing concentrations of BCNU and expressed as percent survival (mean ± SD, n = 6). Significantly different from 9L-S at **P ≤ 0.01 and ****P ≤ 0.0001. BCNU: 1,3-bis (2-chloroethyl)-1-nitrosourea

Figure 2

Dichloroacetate (DCA) improves the responsiveness of resistant glioma cells to BCNU. Treatment of BCNU-sensitive (9L-S) and BCNU-resistant (9L-R) glioma cells with (A) 75 µM or (B) 100 µM BCNU resulted in significant death of 9L-S, but not 9L-R cells. Addition of 25 mM DCA potentiated the action of BCNU in 9L-R cells at both concentrations of BCNU. Bars (mean ± SD, n = 6) with different letters (a vs. b) are significantly different from each other at P ≤ 0.05. DCA: dichloroacetate

Figure 3

Effect of chemoresistance on aspartate isotopomer ratios. A: Model depicting the pathways for aspartate M2 and M3 isotopomer production from U-¹³C glucose; B: Aspartate M3:M2 isotopomer ratios in BCNU-sensitive (9L-S) and BCNU-resistant (9L-R) glioma cells in the absence and presence of 40 mM DCA. Bars (mean ± SD, n = 6) with different letters are significantly different from each other at P ≤ 0.05. PC: pyruvate carboxylase; PDH: pyruvate dehydrogenase; ME: malic enzyme

Figure 4

Effect of chemoresistance on fumarate and succinate isotopomer ratios. Isotopomer ratios for (A) fumarate and (B) succinate in BCNU-sensitive (9L-S) and BCNU-resistant (9L-R) glioma cells in the absence and presence of 40 mM DCA. Bars (mean ± SD) with different letters are significantly different from each other at P ≤ 0.05

Figure 5

Effect of chemoresistance on serine isotopomer ratios. A: Model depicting the pathway for serine M2 and M3 isotopomer production from U-¹³C glucose; B: Serine M3:M2 isotopomer ratios in BCNU-sensitive (9L-S) and BCNU-resistant (9L-R) glioma cells in the absence and presence of 40 mM DCA. Bars (mean ± SD) with different letters are significantly different from each other at P ≤ 0.05

Figure 6

Effect of chemoresistance on glycine isotopomer ratios. A: Model depicting the pathway for glycine M1 and M2 isotopomer production from U-¹³C glucose; B: Glycine M1:M2 isotopomer ratios in BCNU-sensitive (9L-S) and BCNU-resistant (9L-R) glioma cells in the absence and presence of 40 mM DCA. Bars (mean ± SD) with different letters are significantly different from each other at P ≤ 0.05

Figure 7

Effect of BCNU exposure to 9L rat glioma cells. Cells were exposed to 150 µM (9L-R1) or 225 µM (9L-R2) in culture or in vivo (9L-R3) after intracranial implantation (26.6 mg/kg body weight). RNA was subjected to 12-plex microarray to determine changes in gene expression compared to drug naïve cells (9L-S). Venn diagram illustrates the number of genes differentially regulated (P ≤ 0.05) in each of the three BCNU-resistant cell lines