The ketogenic diet and hyperbaric oxygen therapy prolong survival in mice with systemic metastatic cancer

Abstract

Introduction: Abnormal cancer metabolism creates a glycolytic-dependency which can be exploited by lowering glucose availability to the tumor. The ketogenic diet (KD) is a low carbohydrate, high fat diet which decreases blood glucose and elevates blood ketones and has been shown to slow cancer progression in animals and humans. Abnormal tumor vasculature creates hypoxic pockets which promote cancer progression and further increase the glycolytic-dependency of cancers. Hyperbaric oxygen therapy (HBO₂T) saturates tumors with oxygen, reversing the cancer promoting effects of tumor hypoxia. Since these non-toxic therapies exploit overlapping metabolic deficiencies of cancer, we tested their combined effects on cancer progression in a natural model of metastatic disease.

Methods: We used the firefly luciferase-tagged VM-M3 mouse model of metastatic cancer to compare tumor progression and survival in mice fed standard or KD ad libitum with or without HBO₂T (2.5 ATM absolute, 90 min, 3x/week). Tumor growth was monitored by in vivo bioluminescent imaging.

Results: KD alone significantly decreased blood glucose, slowed tumor growth, and increased mean survival time by 56.7% in mice with systemic metastatic cancer. While HBO₂T alone did not influence cancer progression, combining the KD with HBO₂T elicited a significant decrease in blood glucose, tumor growth rate, and 77.9% increase in mean survival time compared to controls.

Conclusions: KD and HBO₂T produce significant anti-cancer effects when combined in a natural model of systemic metastatic cancer. Our evidence suggests that these therapies should be further investigated as potential non-toxic treatments or adjuvant therapies to standard care for patients with systemic metastatic disease.

Figure 1. The KD and HBO₂T increases survival time in mice with systemic metastatic cancer.

(A) Kaplan-Meier survival plot of study groups. Animals receiving KD and KD+HBO₂T showed significantly longer survival compared to control animals (p = 0.0194 and p = 0.0035, respectively; Kaplan-Meier and LogRank Tests for survival distribution). (B) Treatment group cohort size and mean survival times shown. KD mice exhibited a 56.7% increase in mean survival time compared to controls (p = 0.0044; two-tailed student’s t-test); KD+HBO₂T mice exhibited a 77.9% increase in mean survival time compared to controls (p = 0.0050; two-tailed student’s t-test). Results were considered significant when p<0.05.

Figure 2. Tumor bioluminescence in mice.

Tumor growth was slower in mice fed the KD than in mice fed the SD. (A) Representative animals from each treatment group demonstrating tumor bioluminescence at day 21 after tumor cell inoculation. Treated animals showed less bioluminescence than controls with KD+HBO2T mice exhibiting a profound decrease in tumor bioluminescence compared to all groups. (B) Total body bioluminescence was measured weekly as a measure of tumor size; error bars represent ±SEM. KD+HBO2T mice exhibited significantly less tumor bioluminescence than control animals at week 3 (p = 0.0062; two-tailed student‚s t-test) and an overall trend of notably slower tumor growth than controls and other treated animals throughout the study. (C,D) Day 21 ex vivo organ bioluminescence of SD and KD+HBO2T animals (N = 8) demonstrated a trend of reduced metastatic tumor burden in animals receiving the combined therapy. Spleen bioluminescence was significantly decreased in KD+HBO2T mice (*p = 0.0266; two-tailed student‚s t-test). Results were considered significant when p<0.05.

Figure 3. Blood glucose and β-hydroxybutyrate levels in animals.

(A) KD-fed mice showed lower blood glucose than controls on day 7 (***p<0.001). Animals in the KD study group had significantly lower blood glucose levels than controls on day 14 (*p<0.05). (B) KD+HBO2T mice had significantly higher blood ketones than controls on day 7 (***p<0.001). Error bars represent ±SEM. Blood analysis was performed with One Way ANOVA with Kruksal Wallis Test and Dunn‚s Multiple Comparison Test post hoc; results were considered significant when p<0.05.

Figure 4. Animal weight.

Body weight was measured twice a week. Graph indicates average percent of initial body weight animals at days 7 and 14. KD and KD+HBO2T mice lost approximately 10% of their body weight by day 7 and exhibited a significant difference in percent body weight change compared to control animals (*p<0.05; ***p<0.001). Error bars represent ±SEM.

Figure 5. Glucose and weight change are correlated to survival.

Linear regression analysis revealed a significant correlation between day 7 blood glucose and percent body weight change with survival (p = 0.0189 and p = 0.0001, respectively). Results were considered significant when p<0.05.