Evaluation of Temozolomide and Fingolimod Treatments in Glioblastoma Preclinical Models

Abstract

Glioblastomas are malignant brain tumors which remain lethal due to their aggressive and invasive nature. The standard treatment combines surgical resection, radiotherapy, and chemotherapy using Temozolomide, albeit with a minor impact on patient prognosis (15 months median survival). New therapies evaluated in preclinical translational models are therefore still required to improve patient survival and quality of life. In this preclinical study, we evaluated the effect of Temozolomide in different models of glioblastoma. We also aimed to investigate the efficacy of Fingolimod, an immunomodulatory drug for multiple sclerosis also described as an inhibitor of the sphingosine-1-phosphate (S1P)/S1P receptor axis. The effects of Fingolimod and Temozolomide were analyzed with in vitro 2D and 3D cellular assay and in vivo models using mouse and human glioblastoma cells implanted in immunocompetent or immunodeficient mice, respectively. We demonstrated both in in vitro and in vivo models that Temozolomide has a varied effect depending on the tumor type (i.e., U87MG, U118MG, U138MG, and GL261), demonstrating sensitivity, acquired resistance, and purely resistant tumor phenotypes, as observed in patients. Conversely, Fingolimod only reduced in vitro 2D tumor cell growth and increased cytotoxicity. Indeed, Fingolimod had little or no effect on 3D spheroid cytotoxicity and was devoid of effect on in vivo tumor progression in Temozolomide-sensitive models. These results suggest that the efficacy of Fingolimod is dependent on the glioblastoma tumor microenvironment. Globally, our data suggest that the response to Temozolomide varies depending on the cancer model, consistent with its clinical activity, whereas the potential activity of Fingolimod may merit further evaluation.

Keywords: Fingolimod; Temozolomide; brain cancer; glioblastoma; glioma; preclinical models; tumor progression.

Figure 1

Effects of Temozolomide (TMZ) on glioblastoma cells in 3D tumor spheroid assay. (A–F) Dose–response effect of TMZ treatment on U87MG (A–C) and U118MG (D–F) spheroid size (A,D) and cytotoxicity (B,E). The spheroid area (red line) and fluorescent-positive surface (blue line) were quantified per spheroid (C,F). Representative pictures of cells after 69 h of post-treatment with 0, 100, and 1000 µM of TMZ are shown. Scale = 500 µm. The assay was performed with 3 to 4 replicates from 2 to 3 independent experiments. Data represent the mean and SEM. Statistical differences were determined using a mixed-effects model (REML, groups and time as factor) and Bonferroni’s multiple comparisons test (vs. control, * p  ≤  0.05).

Figure 2

Effects of Temozolomide (TMZ) treatment in glioblastoma mouse subcutaneous graft models. (A–D) Impact of TMZ at 10 mg/kg administered 5 times a week (p.o.) on tumor volume in GL261 allograft (A), U87MG xenograft (B), U118MG xenograft (C), and U138MG xenograft (D) models. Discontinuous line highlights treatment beginning. Statistical differences between the groups were determined using a mixed-effects model (REML, groups and time as factor) followed by Bonferroni’s multiple comparisons test (* p  ≤  0.05). Data represent mean and SD. n = 10 (A), n = 6 (B), n = 7 (C), and n = 8 (D) mice per group at the start of treatments.

Figure 3

Effects of Temozolomide (TMZ) treatment in the GL261 glioblastoma mouse orthotopic allograft model. (A–C) Impact of TMZ at 10 mg/kg administered 5 times a week (p.o.) on tumor volume (A), body weight (B), and survival (C). Discontinuous line highlights treatment beginning. Statistical differences between the groups were determined using a mixed-effects model (REML, groups and time as factor) followed by Bonferroni’s multiple comparisons test (* p  ≤  0.05, ** p  ≤  0.01, **** p  ≤  0.001, control vs. all other condition; $ p  ≤  0.05, TMZ 10 mg/kg vs. sham and naïve). Data represent mean and SD. n = 8 mice per group at the start of treatments. For body weight, data reporting was stopped when the first mouse per group had to be sacrificed.

Figure 4

Effects of Temozolomide (TMZ) treatment in the U87MG-RFP-Luc glioblastoma mouse orthotopic xenograft model. (A–C) Impact of TMZ at 10 mg/kg administered 5 times a week (p.o.) on tumor volume measured by bioluminescence imaging (A), body weight (B), and survival (C). Discontinuous line highlights treatment beginning. Statistical differences between the groups were determined using a mixed-effects model (REML, groups and time as factor) followed by Bonferroni’s multiple comparisons test (* p  ≤  0.05, control vs. all other condition; $ p  ≤  0.05, TMZ 10 mg/kg vs. Sham). Data represent mean and SD. n = 8 mice per group at the start of treatments. For body weight, data reporting was stopped when the first mouse per group had to be sacrificed.

Figure 5

Analysis of publicly available clinical data of S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5 expression in GBM patients from the TCGA cohort. (A–E) S1PR2 and S1PR3 are significantly overexpressed in GBM tumors as compared to non-tumoral brains. S1PR1, S1PR4, and SP1R5 are not significantly affected despite an observable increase for S1PR4. The number of patients in each group is indicated below the graphs (T = tumor, in red; N = normal non-tumoral brain, in grey). The difference in expression level between the groups of individuals was evaluated by ANOVA. * p  ≤  0.05 (F–J) High expression of S1PR4 is correlated with more limited patient survival. Expression of S1PR1, S1PR2, S1PR3, and S1PR5 did not correlate with patient survival. Patients were stratified through high and low expression of S1PRs using the median as the cut-off value. The number of patients in each group is indicated. The statistical difference in survival was evaluated using the log-rank test.

Figure 6

Effects of Fingolimod on GBM cells in 3D tumor spheroid assay. (A–F) Dose–response effect of Fingolimod treatment on U87MG (A–C) and GL261 (D–F) spheroid size (A,D) and cytotoxicity (B,E). The spheroid area and fluorescent-positive surface were quantified per spheroid. Representative pictures of cells after 69 h of post-treatment with 0, 5, and 10 µM of Fingolimod are shown. Scale = 500 µm. The assay was performed with 3 to 4 replicates from 2 to 3 independent experiments. Data represent mean and SEM. Statistical differences were determined using a mixed-effects model (REML, groups and time as factor) and Bonferroni’s multiple comparisons test (vs. control, * p  ≤  0.05).

Figure 7

Effects of Temozolomide (TMZ) and Fingolimod treatments in the GL261 glioma mouse orthotopic allograft model. (A–D) Impact of TMZ at 1 and 10 mg/kg, Fingolimod at 5 mg/kg, or a combination administered 5 times a week (p.o.) on tumor volume measured by bioluminescence imaging, including representative images (A), body weight (B), neuroscore (C), and survival (D). Discontinuous line highlights treatment beginning. Statistical differences between the groups were determined using a mixed-effects model (REML, groups and time as factor) followed by Tukey’s multiple comparisons test # p  ≤  0.05, TMZ 10 mg/kg vs. Control or TMZ 1 mg/kg or TMZ 1 mg/kg + Fingolimod 5 mg/kg or Fingolimod 5 mg/kg; $ p  ≤  0.05, Sham vs. Control or TMZ 1 mg/kg or TMZ 1 mg/kg + Fingolimod 5 mg/kg or Fingolimod 5 mg/kg). Data represent mean and SD. n = 8 mice per group at the start of treatments.

Figure 8

Effects of Temozolomide (TMZ) and Fingolimod treatments in the U87MG human orthotopic xenograft model. (A–D) Impact of TMZ at 1 and 10 mg/kg, Fingolimod at 5 mg/kg, or a combination administered 5 times a week (p.o.) on tumor volume measured by bioluminescence imaging, including representative images (A), body weight (B), neuroscore (C), and survival (D). Discontinuous line highlights treatment beginning. Statistical differences between the groups were determined using a mixed-effects model (REML, groups and time as factor) followed by Tukey’s multiple comparisons test (* p  ≤  0.05, vs. Control; $ p  ≤  0.05, Fingolimod 5 mg/kg vs. TMZ 1 mg/kg or TMZ 1 mg/kg + Fingolimod 5 mg/kg; & p  ≤  0.05, Fingolimod 5 mg/kg vs. Sham; £ p  ≤  0.05, Fingolimod 5 mg/kg vs. TMZ 1 mg/kg or TMZ 1 mg/kg + Fingolimod 5 mg/kg or TMZ 10 mg/kg or Sham; # p  ≤  0.05, TMZ 10 mg/kg vs. TMZ 1 mg/kg or TMZ 1 mg/kg + Fingolimod 5 mg/kg; ¤ p  ≤  0.05, Control vs. TMZ 10 mg/kg or TMZ 1 mg/kg or TMZ 1 mg/kg or TMZ 1 mg/kg + Fingolimod 5 mg/kg or Sham; § p  ≤  0.05, Fingolimod 5 mg/kg vs. TMZ 10 mg/kg or TMZ 1 mg/kg or TMZ 1 mg/kg or TMZ 1 mg/kg + Fingolimod 5 mg/kg or Sham). Data represent mean and SD. n = 6–8 mice per group at the start of treatments.