Radiation Induced Metabolic Alterations Associate With Tumor Aggressiveness and Poor Outcome in Glioblastoma

Abstract

Glioblastoma (GBM) is uniformly fatal with a 1-year median survival, despite best available treatment, including radiotherapy (RT). Impacts of prior RT on tumor recurrence are poorly understood but may increase tumor aggressiveness. Metabolic changes have been investigated in radiation-induced brain injury; however, the tumor-promoting effect following prior radiation is lacking. Since RT is vital to GBM management, we quantified tumor-promoting effects of prior RT on patient-derived intracranial GBM xenografts and characterized metabolic alterations associated with the protumorigenic microenvironment. Human xenografts (GBM143) were implanted into nude mice 24 hrs following 20 Gy cranial radiation vs. sham animals. Tumors in pre-radiated mice were more proliferative and more infiltrative, yielding faster mortality (p < 0.0001). Histologic evaluation of tumor associated macrophage/microglia (TAMs) revealed cells with a more fully activated ameboid morphology in pre-radiated animals. Microdialyzates from radiated brain at the margin of tumor infiltration contralateral to the site of implantation were analyzed by unsupervised liquid chromatography-mass spectrometry (LC-MS). In pre-radiated animals, metabolites known to be associated with tumor progression (i.e., modified nucleotides and polyols) were identified. Whole-tissue metabolomic analysis of pre-radiated brain microenvironment for metabolic alterations in a separate cohort of nude mice using ¹H-NMR revealed a significant decrease in levels of antioxidants (glutathione (GSH) and ascorbate (ASC)), NAD⁺, Tricarboxylic acid cycle (TCA) intermediates, and rise in energy carriers (ATP, GTP). GSH and ASC showed highest Variable Importance on Projection prediction (VIPpred) (1.65) in Orthogonal Partial least square Discriminant Analysis (OPLS-DA); Ascorbate catabolism was identified by GC-MS. To assess longevity of radiation effects, we compared survival with implantation occurring 2 months vs. 24 hrs following radiation, finding worse survival in animals implanted at 2 months. These radiation-induced alterations are consistent with a chronic disease-like microenvironment characterized by reduced levels of antioxidants and NAD⁺, and elevated extracellular ATP and GTP serving as chemoattractants, promoting cell motility and vesicular secretion with decreased levels of GSH and ASC exacerbating oxidative stress. Taken together, these data suggest IR induces tumor-permissive changes in the microenvironment with metabolomic alterations that may facilitate tumor aggressiveness with important implications for recurrent glioblastoma. Harnessing these metabolomic insights may provide opportunities to attenuate RT-associated aggressiveness of recurrent GBM.

Keywords: glioblastoma (GBM); metabolomics; radiation therapy (RT); recurrence; tumor microenvironment (TME).

Figure 1

(A) Scheme of experiments. (B) Representative image (at 10X, transmitted light microscopy) of GBM143 xenograft line cultured for 3 weeks in vitro in media indicated; cells were collected and orthotopically implanted into cranially irradiated mice 24 hrs post-irradiation (IR). (C) Representative Immunofluorescence (IF) images (at 4X, tiling) for hLamin A+C staining from 0 Gy and 20 Gy-IR mice coronal sections to assess tumor growth and invasion. (D) Top: Representative images (at 20X) show IF staining at tumor; and the dot-plot of single cell count for hLamin A+C and Ki67 staining. Bottom: Representative images (at 20X) show IF staining at center of corpus callosum, and dot-plot of single cell count for hLamin A+C. IH, ipsilateral hemisphere; CH, contralateral hemisphere; IR, irradiation. Statistical significance is represented as ^*p < 0.05.

Figure 2

Metabolomics of pre-radiated brain using ¹H-NMR: (A) (i) Principal component analysis (PCA) between athymic nude mice groups, 0 Gy and 20 Gy. Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) shows further separation of 20 Gy mice group from non-irradiated control based on differences in the metabolite composition of groups with predicted-variable importance in the projection values shown in graph below. (ii) The graphs show, significantly altered metabolites between the 0 Gy vs. 20 Gy. (B) Multivariate analysis of NMR data performed using SIMCA 15 software (Sartorius Stedim Biotech, Göttingen, German) for cohort of C57BL/6 mice, having groups as indicated. Supervised Partial Least Squares discriminant analysis (PLS-DA) performed shows, separation of all three groups. Bar graphs show metabolites most significantly altered between groups. Additional graphs for metabolic variants observed in C57BL/6 mice are included in Figure S3. Statistical significance is represented as ^*p < 0.05; ^**p < 0.01.

Figure 3

Metabolomics of pre-radiated brain using GC-MS: (A) The graphs show significantly altered metabolites between athymic nude mice groups, 0 Gy and 20 Gy. (B) The graphs show significantly altered metabolites between C57BL/6 mice grouped 0, 20, and 4 Gy × 10. Additional graphs for metabolic variants in C57BL/6 mice groups are in Figure S4. Heatmaps for GC-MS are included in Figure S2C. (C) General scheme for ascorbate (ASC) and glutathione (GSH) cycle in clearance of reactive oxygen species (ROS). Intermediates of ascorbic acid catabolism are represented in orange boxes. Reactions in green show ASC-dependent peroxide metabolism; reactions in the central gray box show GSH-dependent regeneration of ASC; and reactions in red show GSH-dependent peroxide metabolism. Box on the right illustrates the expected metabolic alterations upon irradiation, which include increases in levels of ROS, and utilization of GSH and ASC, with concomitant increase in by-products of ASC catabolism, Threonic acid (ThrO), and Oxalic acid (OxA). Key to metabolic cycle illustrated: ASC, Ascorbate; MDHA, Monodehydroascorbate; MDHAR, Monodehydroascorbate reductase; APX, ASC peroxidase; GR, GSH reductase; GRX, Glutaredoxin; PRX, Peroxiredoxin; ThrO, L-threonic acid; OxA, oxalic acid; DHA, Dehydroascorbic acid; GSH, Glutathione reduced; GSSG, Glutathione, oxidized; NADP⁺, Nicotinamide adenine dinucleotide phosphate. Statistical significance is represented as ^*p < 0.05; ^**p < 0.01.

Figure 4

Immunostaining for microglia, with Iba-1: (A) Immunofluorescence (IF) Images for microglial staining (Iba1/DAPI) and morphology (at 20X), in ipsilateral (IH) and contralateral (CH) hemispheres of 0 Gy and 20 Gy mice injected with GBM143 PDX line. (B) the IF images skeletonized using Image J software to assess microglial morphology. (C) the microglial staining in ipsilateral hemispheres of 0 Gy-GBM143, and 2 Gy-GBM143 compared with that of ipsilateral hemispheres of two separate mice cranially irradiated with 20 Gy-single dose; however, not injected with any human-GBM PDX line. (D) (i) Site of GBM143 injection at IH of mice having received ± cranial irradiation (ii) Stages of microglial activation observed in experimental setting.

Figure 5

Effects of radiation-associated metabolic alteration on GBM outcome: (A) Survival curves: Graphs show difference between survival of irradiated (IR) mice cohorts injected with GBM143 24 hrs post-irradiation (short-term IR, ST-IR) or 2 months post-irradiation (long-term IR, LT-IR). Statistical significance is represented as ^**p < 0.01, ^****p < 0.0001. (B) Proposed model for radiation associated metabolic alterations and their effects on cell processes and glioblastoma multiforme (GBM) outcome. The box marked with yellow outline (irradiated by RT) shows metabolic changes in the radiated brain stromal microenvironment, with rise in energy carriers ATP and GTP, and reduction in levels of antioxidants, ascorbate and glutathione, and the cellular processes affected by them. With IR, continued and excess rise in levels of energy carriers and expense of antioxidants within stromal cells of the brain can lead to altered extracellular milieu. Pathophysiological changes in the extracellular milieu, which can be immediate or long term caused by the radiation are enlisted in purple box. These alterations would collectively contribute to radiated stroma and GBM cell interactions that are permissive to GBM growth and, aggressive recurrence. Translational relevance of the study and its insights gained from pre-radiated brain microenvironment to prevent secondary and recurrent GBM spread is illustrated in Figure S5.