Changes in the tumor microenvironment and outcome for TME-targeting therapy in glioblastoma: A pilot study

Abstract

Glioblastoma (GBM) is a hypervascular and aggressive primary malignant tumor of the central nervous system. Recent investigations showed that traditional therapies along with antiangiogenic therapies failed due to the development of post-therapy resistance and recurrence. Previous investigations showed that there were changes in the cellular and metabolic compositions in the tumor microenvironment (TME). It can be said that tumor cell-directed therapies are ineffective and rethinking is needed how to treat GBM. It is hypothesized that the composition of TME-associated cells will be different based on the therapy and therapeutic agents, and TME-targeting therapy will be better to decrease recurrence and improve survival. Therefore, the purpose of this study is to determine the changes in the TME in respect of T-cell population, M1 and M2 macrophage polarization status, and MDSC population following different treatments in a syngeneic model of GBM. In addition to these parameters, tumor growth and survival were also studied following different treatments. The results showed that changes in the TME-associated cells were dependent on the therapeutic agents, and the TME-targeting therapy improved the survival of the GBM bearing animals. The current GBM therapies should be revisited to add agents to prevent the accumulation of bone marrow-derived cells in the TME or to prevent the effect of immune-suppressive myeloid cells in causing alternative neovascularization, the revival of glioma stem cells, and recurrence. Instead of concurrent therapy, a sequential strategy would be better to target TME-associated cells.

Fig 1. CSF1R conditional knockout mouse and GBM development.

(A) Agarose gel electrophoresis showing homozygous CSF1R^flox/flox /MX1-Cre+ (knockout) genotype. (B) Violin plot showing flow-cytometric analysis of peripheral blood cells from conditional knock out mice showed a significant dose-dependent decrease in CD45+CSF1R+ cells following two weeks of treatments with poly-IC. n = 8. (C) Violin plot showing flow-cytometric analysis of peripheral blood cells from wild type mice did not show any significant difference in CD45+CSF1R+ cells following two weeks of treatments with poly-IC. n = 6.

Fig 2

(A) Schematic representation of the study design and treatment schedule. (B) No significant difference in average bodyweights before and after the implantation of the tumor was observed over the period of 2 weeks.

Fig 3

(A and B) Optical images and quantified photon intensities of pre and post-treatment (either vehicle or SB225002) showed significantly increased tumor growth in the vehicle-treated wild-type animals after 3 weeks. Knockout (KO) animals treated with either vehicle or SB225002 and wild-type animals treated with SB225002 (SB) did not show any significant tumor growth after 3 weeks. (C) Flow-cytometric analysis showing a significant decrease in CSF1R+ cells in brain tumor (left panel) and spleen (right panel) of the Knockout mice compared to the wild type mice treated with vehicle. Quantitative data are expressed in mean ± SEM. *P < .05, **P < .01, ****P < .0001. n = 4.

Fig 4. Flowcytometric analysis of myeloid cell and tumor-associated macrophage populations in wild type and KO animals treated with either vehicle or SB225002 (SB).

Both (A) myeloid cells (CD45+CD11b+) and (B) TAMs (CD45+CD11b+CD206+) cells were significantly decreased in the brain (left panel) and spleen (right panel) of the KO mice while a decline was more prominent in the KO mice treated with SB225002. Quantitative data are expressed in mean ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001. n = 3.

Fig 5. Flow cytometric analysis of T-cells and myeloid cell populations in wild type and knockout animals.

There was a significant increase in CD4, CD8, CD11b, and Ly6G positive cells in tumors treated with TMZ, while irradiation caused a significant reduction in different cellular populations compared to the control group. All other treatments showed increased infiltration of CD4 and CD8 T-cells but insignificant changes in MDSCs, CD11b populations. Quantitative data presented in mean ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001, n = 6. TMZ = temozolomide, PD 1 = programmed cells death protein 1, KD = Knockdown.

Fig 6. Flowcytometric analysis of M1 and M2 macrophage populations.

Treatment with TMZ and Vatalanib increased the macrophage population significantly, and all other treatments changed the macrophage population inconsequentially. Quantitative data presented in mean ± SEM. **P < .01, ***P < .001, n = 6. TMZ = temozolomide, PD 1 = programmed cells death protein 1, KD = Knockdown.

Fig 7. Bioluminescent image-based analysis of tumor growth.

All animals underwent optical imaging to monitor tumor growth before starting the treatment (day 8 post-inoculation), 1 week, and 2 weeks after treatment. There was no significant difference between all treatment groups compared to that of vehicle-treated animals after 1 week of treatment except the Vatalanib treated group that showed significant tumor growth. Following 2 weeks of treatment, tumor growths were substantially increased in the vehicle, Vatalanib, and TMZ treated animals. All other groups showed increased tumor growth but were significantly slower than the above-mentioned groups. Quantitative data are expressed in mean ± SEM. *P < .05, **P < .01. n = 3.

Fig 8. Survival studies showing improved survival following the use of TME targeting agents.

(A and B) Kaplan-Meier curve showing significantly increased survival in animal groups treated with TMZ (50mg/kg/day, 3days/week), HET0016 (HET, 10mg/kg/day, 5days/week), TMZ+HET0016, and with a HET analog (10mg/kg/day, 5days/week). Although Navarixin (10mg/kg/day, 5 days/week) increased survival, the addition of TMZ with it did not improve the outcome. A Log-rank test (Mantel-Cox) was applied to determine the significance of differences among the groups. *P < .05, **P < .01. n = 3–5. Dose of Fluoxetine was 10 mg/kg/day, 3days/week. Dose of SB225002 was 10mg/kg/day, 5 days/week. Only wild type animals were used in survival studies.