This study documents the therapeutic action of glucose and glutamine targeting in the VM-M3 and CT-2A syngeneic mouse models of GBM. It is known that calorie restriction, ketogenic diets, and DON can reduce metabolites in the glycolytic and glutaminolysis pathways, respectively33,39,40,41. However, we showed that the KD-R + DON could rescue mice with these GBM tumours from late-stage orthotopic growth. The diet/drug therapy reduced brain swelling, hemorrhage, and morbidity. The KD-R alone reduced the VM-M3/Fluc cell invasion and proliferation as seen by both in vivo and ex vivo bioluminescent imaging of the brains, with Ki-67 staining, and with histology of the brain tissues. Moreover, the KD-R produced therapeutic ketosis (low GKI value) and reduced TNF-α and Iba-1 levels, suggesting reduced inflammation as compared to the SD-UR control brains. The ability of DON treatment to arrest tumour growth and promote survival late in disease progression suggests a potentially direct role for glutamine metabolism in VM-M3/Fluc growth and invasion. This finding is consistent with the importance of glutamine in the metabolism of mesenchymal cells from which the VM-M3 tumour is derived4,37,42. Although DON treatment alone was effective in reducing tumour growth and invasion in the control SD-UR mice, a synergistic effect was observed when DON was administered together with the KD-R, providing evidence for a diet/drug interaction. The findings also support the press-pulse therapeutic strategy for the metabolic management of cancer with the KD-R serving as a press therapy and DON serving as a pulse therapy6.

Interestingly, the level of DON in the brain tumour tissue was higher under KD-R feeding than under SD-UR feeding suggesting that the KD-R facilitates DON delivery to the brain. This is significant because it allowed for a lower therapeutic dose of DON when administered under a KD-R regimen. A similar observation was made previously in showing that a KD-R facilitated the delivery of the iminosugar, N-butyldeoxynojirimycin, across the blood–brain barrier in mice with Sandhoff disease43. Although the mechanism remains to be determined, these findings suggest that the KD has potential as a facilitator of drug delivery to the brain for a broad range of neuro-pathological conditions. It is also important to mention that our previous findings clearly demonstrated that caloric restriction can maintain the blood vessel integrity in the mouse CT-2A and the human U-87 GBM and thus reduce the leakiness of the neovasculature44. Interestingly, a recent observation by Gordon et al. showed that DON treatment promotes the recovery of blood–brain barrier integrity in the mouse model of cerebral malaria33. Further studies are needed to clarify the mechanism by which a KD-R facilitates drug delivery to the brain.

DON has shown therapeutic efficacy in human cancers of the blood, colon, and lung, but issues of toxicity were noted in some cases30. No noticeable toxicity was seen in the DON-treated mice until the end of the study when mild muscle loss was observed in some of the mice. It is proposed that issues with DON toxicity arise when there is an appreciable competition for glutamine between host and tumour. There is also increasing evidence supporting the protective role of glutamine supplementation for cancer patients45. Since tumour consumption of glutamine is dissipative, glutamine supplementation might not increase tumour growth46. However, additional preclinical studies will be needed to determine if glutamine supplementation could be used together with DON. It is our view that strategies for managing toxicity of a therapeutically effective drug would be as important as developing a new drug. Our results indicate that the KD-R enhances the therapeutic action of DON, thus reducing dosage and toxicity.

The therapeutic action of the diet/drug effect seen in the VM-M3 tumour was also seen in the CT-2A neural stem cell tumour. As observed from histological analysis, the KD-R + DON treatment caused massive mitotic arrest or catastrophe in the CT-2A brain tumour cells. The results show that therapeutic efficacy against orthotopic brain tumour growth was greater using the diet/drug combination than in using either the KD-R or DON alone. The CT-2A tumour shares several characteristics with glioma stem cells32, whereas the VM-M3 tumour shares several characteristics with the invasive mesenchymal cell found in most GBM2. Hence, our findings suggest that the KD-R + DON therapeutic strategy could potentially target the two most common neoplastic cell types found in human GBM3. Our recent human GBM case report provides support for our prediction15.

Several mechanisms could underlie the therapeutic action of the diet/drug therapy used to treat the VM-M3 and CT-2A tumours. The KD-R will simultaneously target the glycolytic and pentose phosphate pathways, which are upregulated in GBM47,48,49. We previously showed that calorie restriction and restricted ketogenic diets target the IGF-1, PI3K, AKT, and Hif-1α signaling pathways in the CT-2A tumour38,50. The down-regulation of these pathways would reduce angiogenesis, inflammation, and mTOR signaling, while enhancing tumour cell apoptosis23,44,51,52. As glucose is the fuel for glycolysis, the pentose phosphate pathway, and serine biosynthesis, the KD-R should reduce multiple growth metabolites, tumour cell glutathione levels, and nucleotide synthesis. Furthermore, the KD-R should also reduce one-carbon metabolism especially in glioma cells lacking OxPhos capacity25,49,53. Hence, the glucose-restricting action of the KD-R will target multiple signaling pathways linked to glioma growth and progression.

In addition to restricting glucose availability, the KD-R will also elevate β-hydroxybutyrate and acetoacetate, the major circulating ketone bodies produced in the liver, from the active metabolism of medium chain triglycerides54. Ketone bodies increase the redox span of the CoQ couple, thus reducing oxidative stress in normal brain cells54. These ketone bodies are also neuroprotective and have beneficial therapeutic value for various diseases24,55. As ketone bodies are non-fermentable and require efficient OxPhos for generating ATP25,54, the VM-M3 and CT-2A cells will be unable to metabolize these metabolites for energy due to insufficient OxPhos12,25. Ketone bodies cannot be used as an alternative energy fuel in cells with defective mitochondria12,25. Previous studies demonstrated that glioma cells cannot use ketone bodies and that ketone bodies do not stimulate tumour growth56,57. Hence, the KD-R has a dual function in (1) targeting glucose-dependent signaling pathways that drive tumour growth, and (2) in providing an alternative metabolic fuel to normal brain cells under glucose restriction.

While the KD-R will reduce metabolites through glycolysis and the pentose phosphate pathway, DON will block glutaminolysis, thus depriving the tumour cells of the amide nitrogen needed for nucleotide and protein synthesis, and at the same time, will deplete the glutamate needed for synthesis of α-ketoglutarate (α-KG)25,58,59. α-KG is a precursor for lipid synthesis through reductive carboxylation and is the substrate for ATP synthesis through the succinate-CoA ligase reaction in the TCA cycle under hypoxia25,60. As mentioned previously, however, glutamine targeting is more challenging than is glucose targeting due to the importance of glutamine for the immune system and the gut6,61. Consequently, glutamine targeting must be done strategically to avoid damage to those systems that are needed for normal physiological function6,15. It is for this reason that we administered DON to the tumour-bearing mice at low doses and transiently. We administered DON 7 days after tumour implantation, which is considered late for the VM-M3/Fluc tumour because neurological signs and symptoms start to appear 10–12 days post implantation followed by 100% death in 14–18 days. The late administration of DON was chosen to ensure adequately high ketone levels were achieved in order to facilitate neuroprotection and to reduce tumour inflammation. Secondly, the late treatment could serve as a comparable model when treating late-stage GBM patients. A 60–70% reduction in tumour growth was observed when DON was administered at 0.5 mg/kg once or twice per week. In comparison, a near complete resolution of the brain tumour was achieved when 1.0 mg/kg of DON was administered with the same schedule, as seen with bioluminescent imaging. These results are significant because no other clinical or pre-clinical study using DON considered a KD-R as a method for protecting the host from toxicity. Case reports of GBM and other metastatic cancers show the significance of the KD-R when combined with other therapies15,62,63.

Our findings support previous suggestions that reduced glutaminolysis would be a key therapeutic action of DON against the growth of glutamine-dependent tumours, including GBM29,30. Although it is known that DON targets glutaminolysis in tissues30,33,58 and that reduced levels of circulating glucose will reduce glycolysis64,65, we were unable to find consistent brain metabolite changes in pathways of glycolysis and glutaminolysis in a preliminary metabolomic analysis of KD-R + DON-treated VM-M3 mouse brains. Due to the confounding variables in the in vivo environment (dead tumour cells, necrotic tissue, and various types of host infiltrating cells), we believe that an in vitro analysis in defined media could provide a clearer picture of the changes in metabolites of the glucose and glutamine pathways due to DON or other drug treatments in the VM-M3 and CT-2A cells. It will also be important to determine if other glutaminolysis inhibitors, e.g., epigallocatechin-3-gallate (EGCG), bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTEs), and CB-83919,66, can match the therapeutic action of DON against brain cancer when used together with the KD-R. Pretreatment with a ketogenic diet before implementing the standard of care is suggested in order to protect normal brain cells from treatment toxicities22.

It is important to mention that our findings in the VM-M3 and CT-2A syngeneic GBM models might be considered contradictory to some previous studies in GBM patients and in experimental systems67,68,69. Previous studies from Bachoo and colleagues suggest minimal 13C-glutamine anaplerosis in GBM implying that glutamine restriction in this context may not be therapeutic68. As DON inhibits multiple glutaminases, it is possible that DON does not directly impact glutamine anaplerosis in vivo, but acts on other related pathways and systems29,30,33. We recently described how glutamine is the only amino acid not requiring an energy investment to generate ATP through mitochondria substrate level phosphorylation (mSLP)25. Tardito et al. found that under glutamine starvation, intracellular oleate was unaffected, and glucose-dependent glutamate production increased, implying that the contribution of glutamine to growth is largely independent of anaplerosis69. Further, Oizel et al. identified two clusters of GBM that are glutaminehigh and glutaminelow based on glutamine utilization16. These investigators also mentioned that the most aggressive GBM contained mesenchymal cells that utilized the highest levels of glutamine. The VM-M3 cells are highly invasive and of mesenchymal origin2,37. Nevertheless, we use caution in the interpretation of our data, as the metabolic effects of DON’s action in killing the GBM cells were inferred rather than measured directly.

In the present study, we demonstrate the benefits of targeting glutamine under a KD-R for managing experimental GBM. The influence of targeting both glucose and glutamine on the metabolic pathways involved with GBM growth is shown in Fig. 8. This treatment was capable of arresting the growth of tumour cells and in promoting the survival of mice with two different syngeneic GBM tumours grown orthotopically. As the mice used for these studies were not treated with surgery, radiation, or standard chemotherapy, it is unclear if a similar therapeutic response would be seen in GBM patients using this approach together with current standard of care15. Because of the high mortality following a GBM diagnosis, our findings should have clinical implications especially in light of our recent case report15. Furthermore, our studies reveal a potential role for the ketogenic diet in facilitating drug delivery to brain tumours.