Advanced age negatively impacts survival in an experimental brain tumor model

Abstract

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, with an average age of 64 years at the time of diagnosis. To study GBM, a number of mouse brain tumor models have been utilized. In these animal models, subjects tend to range from 6 to 12 weeks of age, which is analogous to that of a human teenager. Here, we examined the impact of age on host immunity and the gene expression associated with immune evasion in immunocompetent mice engrafted with syngeneic intracranial GL261. The data indicate that, in mice with brain tumors, youth conveys an advantage to survival. While age did not affect the tumor-infiltrating T cell phenotype or quantity, we discovered that old mice express higher levels of the immunoevasion enzyme, IDO1, which was decreased by the presence of brain tumor. Interestingly, other genes associated with promoting immunosuppression including CTLA-4, PD-L1 and FoxP3, were unaffected by age. These data highlight the possibility that IDO1 contributes to faster GBM outgrowth with advanced age, providing rationale for future investigation into immunotherapeutic targeting in the future.

Keywords: Cancer immunity; Glioma; Immunosuppression; TDO; Treg; Tryptophan.

Figure 1. The overall survival of young and old mice with brain tumors

2 × 10⁵ GL261 cells were intracranially-injected into 8 week old (black, n=10) or 74 week old (grey, n=12) C57BL/6 mice. The Kaplan-Meier curve represents mouse survival times over a time course of 50 days. * Gehan-Breslow-Wilcoxon test P = 0.0292.

Figure 2. mRNA expression analysis for immunoregulatory genes in young and old mice with brain tumors

RT-PCR quantitative analysis for IDO1, TDO, PD-L1, CTLA-4, FoxP3, and IFN-γ in GL261 cell-based glioma lysates isolated at 3 weeks post-intracranial engraftment in 8 week old (n=8) and 74 week old (n=5) WT mice. ND = Not detectable. Bar graphs shown as mean ± SEM. Significance determined by One-Way ANOVA followed by a Turkey’s post-hoc test (P<0.01).

Figure 3. Tryptophan (TRP) and kynurenine (KYN) levels in young and old mice with brain tumors

(A) Trp and Kyn levels in the serum and brain tumor (BT) were analyzed by HPLC in 8 week old (n=8) and 74 week old (n=5) mice at 3 weeks post-intracranial engraftment. There was no difference in the (B) KYN/TRP ratio. Bar graphs shown as mean ± SEM.

Figure 4. T cell and antigen presenting cell (APC) levels in young and old mice with brain tumors

Eight week old (n=8) and 74 week old (n=5) mice were intracranially-engrafted 2 × 10⁵ GL261 cells. The absolute numbers of (A) CD4⁺ T cells, CD4⁺FoxP3⁺ T cells, CD8⁺ T cells, CD8⁺CD44^high T cells, (B) macrophages, microglia, dendritic cells, and myeloid derived suppressor cells (MDSC) isolated from brain tumor, spleen, and cervical lymph node (cLN) of tumor-bearing mice at 3 weeks post-engraftment. The flow cytometry gating strategy is demonstrated for (C) T cell and (D) APC subpopulations is demonstrated. Bar graphs in A and B are shown as mean ± SEM.