Evaluating the Combined Effect of a Choline Kinase Inhibitor and Temozolomide Therapy in a Mouse Model of Glioblastoma Using 1H MR Spectroscopy and IVIM-DWI

Abstract

This study evaluated the therapeutic efficacy of combining a choline kinase alpha (ChoKα) inhibitor, MN58b, and temozolomide (TMZ) in a syngeneic GL261 glioblastoma (GBM) mouse model. It used MR spectroscopy (MRS) and intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) to assess metabolic and microstructural changes within the tumor. Fifty-two C57BL/6 mice had GL261 cells implanted intracranially and were divided into four groups: saline control, MN58b, TMZ, and MN58b + TMZ (n = 16, 14, 11, and 11, respectively). Treatments were administered for 5 days, starting 10 days post-implantation. MRI scans (T₂-weighted, MRS, IVIM-DWI) were performed at baseline, during treatment (Day 3), and post-treatment (Day 6). The histological analysis evaluated the tumor mitotic index and caspase-3 expression. Combination therapy with MN58b + TMZ significantly reduced tCho/NAA, Lip + Lac/tCr, and mI/tCr, suggesting decreased phosphocholine synthesis and tumor proliferation. IVIM-DWI showed a significant increase in diffusion coefficient (D) values, indicating reduced cell density. Metabolic changes detected by ¹H MRS were observable as early as Day 3 post-treatment initiation, preceding microstructural alterations detected by IVIM-DWI at Day 6. This suggests that MRS biomarkers may serve as early indicators of treatment response, facilitating timely therapeutic decisions. Histology confirmed a significantly lower mitotic index relative to control tumors in the combination treatment group. Significantly prolonged survival with combination therapy was noted relative to other groups. However, the tumor volumes were not significantly different between groups. Combination therapy targeting ChoKα and cellular proliferation with MN58b and TMZ outperformed individual treatments for GBM, warranting further exploration. The integration of MN58b with the current standard of care (TMZ + RT) might further enhance the therapeutic outcomes. MRS and IVIM-DWI demonstrate potential utility as non-invasive imaging markers for treatment monitoring.

Keywords: GBM; IVIM; MR spectroscopy; cancer; glioblastoma; microstructure; treatment response.

FIGURE 1

(A–D) Treatment effect on GL261 GBM growth. (A) T₂‐weighted image showing hyperintense tumor relative to the surrounding tissue. (B) Diffusion‐weighted image (b = 0 s/mm²) showing the high signal intensity of the tumor compared with the rest of the brain. (C) Percent change in tumor volume relative to Day 0 over treatment time for all cohorts. (D) Kaplan–Meier survival curves showing the survival probability for each group. The asterisk indicates a significant difference between groups at p < 0.05.

FIGURE 2

(A–G) T2‐weighted image (A) displaying the voxel position (highlighted region in dashed red) used for MRS acquisition within the tumor region. Representative MR spectra (B–G) from tumor‐bearing mice at baseline (Day 0), Day 3, and Day 6. Example spectra from a control mouse (B–D) and an MN58b + TMZ treated mouse (E–G) are shown. Resonances typically seen in the tumors have been labelled (Lip + Lac, NAA, Glx, tCr, tCho, and mI).

FIGURE 3

(A–E) Percentage change (relative to baseline) in metabolite ratios following drug treatment. (A) tCho/tCr, (B) tCho/NAA, (C) mI/tCr, (D) Lip + Lac/tCr, and (E) Glx/tCr. The asterisk indicates the difference between groups reached a significance level of p < 0.05.

FIGURE 4

(A–D) Representative intravoxel incoherent motion (IVIM) parameter maps illustrating the apparent diffusion coefficient (ADC), pure diffusion coefficient (D), pseudo‐diffusion coefficient (D*), and perfusion fraction (f). The tumor region of interest (ROI) is delineated in magenta.

FIGURE 5

(A–D) Box plots illustrating the percent change in the IVIM‐DWI parameters, ADC, D, D*, and f, with time with respect to Day 0 for all groups. The asterisk indicates the difference between groups where p < 0.05.

FIGURE 6

(A–J) Representative H&E images from the four experimental groups. (A–D) Representative high‐power fields close to the normal brain parenchyma (*) with highlighted mitotic figures (green circles) for saline (A), MN58b (B), TMZ (C), and MN58b + TMZ (D), hematoxylin–eosin, 400×. (E–H) Representative examples of positive cells for caspase‐3 expression (arrowhead) for saline (E), MN58b (F), TMZ (G), and MN58b + TMZ (H). Scalebars: 50 μm. Mitotic index (I) and percentage positive apoptotic cells (J) measurements were assessed from the tumor margins of three animals from each treatment group.