The Ketogenic Diet and Hyperbaric Oxygen Therapy Prolong Survival in Mice with Systemic Metastatic Cancer

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.


Introduction
Metastasis is a complex phenomenon in which cancer cells spread from a primary tumor to establish foci in a distal tissue and is responsible for 90 percent of cancer-related deaths [1]. The specific changes which mediate metastasis remain unclear; however, the process generally involves local tumor growth, invasion through the basement membrane and surrounding tissue, intravasation into the lymphatics or blood vessels, dissemination and survival in circulation, extravasation from the vasculature, and re-establishment of tumors at distal tissues. While many primary tumors can be controlled with conventional therapies like surgery, chemotherapy, and radiation, these treatments are often ineffective against metastatic disease and in some cases may promote cancer progression and metastasis [2,3,4]. There is a substantial need for novel therapies effective against metastatic cancer.
Perhaps the most important limiting factor in the development of new treatments for metastatic cancer is the lack of animal models that accurately reflect the true nature of metastatic disease. Xenograft models of human cancers in immunodeficient mice are inadequate as the immune system is highly involved in cancer development and progression. Indeed, most tumor models grown as xenografts in immune compromised mice fail to metastasize [5,6]. Tail vein injection models of metastatic cancer eliminate the important steps of local tissue invasion and intravasation into the vasculature, again failing to represent the true disease phenotype. The VM-M3 model of metastatic cancer is a novel murine model that closely mimics the natural progression of invasion and metastasis [7,8]. The VM-M3 tumor arose spontaneously in the brain of a mouse of the VM/Dk inbred strain and expresses multiple growth characteristics of human glioblastoma multiforme with macrophage/microglial properties [7,9]. When implanted subcutaneously, VM-M3 cells rapidly metastasize to all major organ systems, notably the liver, lung, kidney, spleen, brain, and bone marrow. Systemic metastasis has also been repeatedly documented in human glioblastoma multiforme (GBM), which has been linked to the macrophage/microglial characteristics of the tumor [9]. The tumor was adapted to cell culture and transfected with the firefly luciferase gene to allow for easy monitoring of tumor growth in vivo [10]. The VM-M3 model of metastatic cancer has a distinct advantage over other metastatic models because it spreads naturally in an immunocompetent host, mimicking the natural cancer microenvironment. Cisplatin and methotrexate, two commonly-used chemotherapy agents, inhibit metastatic spread in the VM-M3 model of metastatic cancer similarly to their effects in humans, further supporting the model's representation of the true disease state [8,11,12,13]. For these reasons, the VM-M3 model of metastatic cancer was used for this study.
Abnormal energy metabolism is a consistent feature of most tumor cells across all tissue types [14]. In the 1930 s, Otto Warburg observed that all cancers expressed high rates of fermentation in the presence of oxygen [15]. This feature, known as The Warburg Effect, is linked to mitochondrial dysfunction and genetic mutations within the cancer cell [14,16,17]. These defects cause cancers to rely heavily on glucose for energy, a quality that underlies the use of fluorodeoxyglucose-PET scans as an important diagnostic tool for oncologists [18]. Ketogenic diets are high fat, low carbohydrate diets that have been used for decades to treat patients with refractory epilepsy [19]. Ketogenic diets also suppress appetite naturally thus producing some body weight loss [19,20,21,22]. Dietary energy reduction (DER) lowers blood glucose levels, limiting the energy supply to cancer cells, while elevating circulating blood ketone levels [6]. Ketone bodies can serve as an alternative energy source for those cells with normal mitochondrial function [23,24], but not for cancer cells [25]. DER has been shown to have anti-tumor effects in a variety of cancers, including brain, prostate, mammary, pancreas, lung, gastric, and colon [14,26,27,28,29,30,31,32,33,34]. DER produces anti-cancer effects through several metabolic pathways, including inhibition of the IGF-1/PI3K/Akt/HIF-1a pathway which is used by cancer cells to promote proliferation and angiogenesis and inhibit apoptosis [35,36,37,38,39,40,41,42]. Additionally, DER induces apoptosis in astrocytoma cells, while protecting normal brain cells from death through activation of adenosine monophosphate kinase (AMPK) [43].
Tumors possess abnormal vasculature which blocks adequate tissue perfusion, leading to the presence of hypoxic regions that promote chemotherapy and radiation resistance [44,45,46,47]. In fact, hypoxic cancer cells are three-times more resistant to radiation therapy than are well-oxygenated cells [48]. In addition to decreasing the efficacy of standard care, tumor hypoxia activates a number of oncogene pathways, largely through the HIF-1 transcription factor, which promote tumor growth, metastasis, angiogenesis, and inhibit apoptosis [49,50].
Hyperbaric oxygen therapy (HBO 2 T) involves administration of 100% oxygen at elevated pressure (greater than sea level, or 1 ATA). HBO 2 T increases plasma oxygen saturation which facilitates oxygen delivery to the tissue independent of hemoglobin O 2 saturation [51]. The potential benefit of using HBO 2 T to combat the cancer-promoting effects of tumor hypoxia is clear. HBO 2 T alone has been shown to inhibit tumor growth, reduce tumor blood vessel density, and induce the preferential expression of anti-cancer genes in rat models of mammary tumors [52]. Additionally, radiation and many chemotherapy drugs work by producing free radicals within the tumors, leading to cell death. HBO 2 T enhances tumor-cell production of reactive oxygen species which contributes to the synergistic effects of HBO 2 T as an adjuvant treatment to standard care. Indeed, HBO 2 T enhances the efficacy of both radiation and chemotherapy in animal models [53,54,55,56,57].
In normal tissues, decreased oxygen availability inhibits mitochondrial production of ATP, stimulating an up-regulation of glycolytic enzymes to meet energy needs by substrate level phosphorylation production of ATP. Thus, the cellular response to tumor hypoxia is mediated by several of the same pathways that are overly active in cancer cells with mitochondrial damage and high rates of aerobic glycolysis. This suggests that the ketogenic diet and HBO 2 T could target several overlapping pathways and tumorigenic behaviors of cancer cells. We hypothesized that these treatments would work synergistically to inhibit tumor progression. We suggest that the addition of these non-toxic adjuvant therapies to the current standard of care may improve progression free survival in patients with advanced metastatic disease.

Mice
Three breeding pairs of the VM/Dk strain of mice were used to establish and propagate a VM/Dk mouse colony in the University of South Florida (USF) Morsani College of Medicine Vivarium according to standard husbandry protocol. Forty adult male mice (10-18 weeks of age) were used for this study. All animal procedures were performed within strict adherence to the NIH Guide for the Care and Use of Laboratory and Animals and were approved by the USF Institutional Animal Care and Use Committee (IACUC; Protocol Number R4137).

Cell Culture
VM-M3/Fluc cells were received as a gift from T.N. Seyfried, Boston College, where they were created from a spontaneous tumor in a VM/Dk mouse and adapted to cell culture [7]. VM-M3/Fluc cells were transduced with a lentivirus vector containing the firefly luciferase gene under control of the cytomegalovirus promoter (VM-M3/Fluc) as previously described [7]. The VM-M3/Fluc cells were cultured in Eagle's Minimum Essential Medium with 2 mM L-glutamine (ATCC, Manassas, VA), 10% fetal bovine serum (Invitrogen, Grand Island, NY), 1% penicillinstreptomycin (Gibco, Invitrogen) and high glucose (25 mM Dglucose, Fisher Scientific, Waltham, MA). Cells were cultured in a CO 2 incubator at 37uC in 95% air and 5% CO 2 .

Subcutaneous Tumor Implantation
On day 0, VM-M3/Fluc cells (1 million cells in 300 mL PBS) were implanted, s.c., into the abdomen of VM/Dk mice using a 27 gage needle. Inoculation results in rapid and systemic metastasis to most major organs, namely liver, kidneys, spleen, lungs, and brain as previously described [7].

Diet Therapy
On the day of tumor inoculation, mice were randomly assigned to one of four groups: SD (Control); SD+HBO 2 T; KD; or KD+HBO 2 T. Mice in the SD group were fed standard rodent chow (2018 Teklad Global 18% Protein Rodent Diet, Harlan) ad libitum. Mice in the KD group received KD-Solace ketogenic diet ad libitum. KD-Solace is a commercially available ketogenic diet powder (KetoGen, Solace Nutrition) and was mixed 1:1 with H 2 O to form a solid paste. Macronutrient information for SD and KD-Solace are shown in Table 1. Diets were continuously replaced every other day to maintain freshness and allow mice to feed ad libitum.

Glucose, Ketone, and Weight Measurements
Every 7 days, blood was collected from the tail using approved methods. Glucose was measured using the Nova MaxH Plus TM Glucose and b-Ketone Monitoring System (Nova Biomedical, Waltham, MA), and b-hydroxybutyrate was measured using the Precision Xtra TM Blood Glucose & Ketone Monitoring System (Abbott Laboratories, Abbott Park, IL).
Mice were weighed between 1 and 3 pm twice a week for the duration of the study using the AWS-1KG Portable Digital Scale (AWS, Charleston, SC).

Bioluminescent Imaging and Tumor Growth Analysis
Tumor growth was monitored as a measure of bioluminescent signaling using the Xenogen IVIS Lumina system (Caliper LS, Hopkinton, MA). Data acquisition and analysis was performed using the Living ImageH software (Caliper LS). Approximately 15 minutes prior to in vivo imaging, the mice received an i.p. injection of D-Luciferin (50 mg/kg) (Caliper LS). Bioluminescent signal was obtained using the IVIS Lumina cooled CCD camera system with a 1 sec exposure time. As only the cancer cells contained the luciferase gene, bioluminescent signal (photons/sec) of the whole animal was measured and tracked over time as an indicator of metastatic tumor size and spread.

Survival Analysis
Throughout the study, health and behavior of the mice were assessed daily. Mice were humanely euthanized by CO 2 asphyxiation according to IACUC guidelines upon presentation of defined criteria (tumor-associated ascites, diminished response to stimuli, lethargy, and failure to thrive), and survival time was recorded.

Statistics
Survival data was analyzed by the Kaplan-Meier and Logrank Tests for survival distribution. Mean survival times were analyzed by two-tailed student's t-tests. Bioluminescent signal as a measure of tumor size was analyzed by two-tailed student's t-tests. Blood measurements were analyzed by One Way ANOVA with Kruskal Wallis Test and Dunn's Multiple Comparison Test post hoc. Differences in percent weight change were analyzed by One Way ANOVA with Tukey's Multiple Comparison Test post hoc. Correlation between blood glucose, body weight change, and survival times were analyzed by linear regression analysis. Results were considered significant when p,0.05.

Results
Combining the KD with HBO 2 T Prolonged Survival in Mice with Metastatic Cancer KD and KD+HBO 2 T treated mice demonstrated a statistically different survival curve by Logrank Test with an increase in survival time compared to control animals (p = 0.0194 and p = 0.0035, respectively; Figure 1A). KD fed and KD+HBO 2 T animals also showed a significant increase in mean survival time compared to control animals by the two-tailed student's t-test (p = 0.0044 and p = 0.0050, respectively; Figure 1B). Although previous studies have reported that HBO 2 T alone can increase survival time in animals with various cancers [52,54,58,59], we did not see an effect on survival in mice receiving SD+HBO 2 T. Control (SD) mice had a mean survival time of 31.2 days whereas SD+HBO 2 T mice had a non-statistically different mean survival of 38.8 days ( Figure 1B). The KD alone increased mean survival time by approximately 17 days (56.7%), and when combined with HBO 2 T, mice exhibited an increase in mean survival time of approximately 24 days (77.9%) ( Figure 1B). This finding strongly supports the efficacy of the KD and HBO 2 T as therapies to inhibit tumor progression and prolong survival in animals with metastatic cancer.

The KD and HBO 2 T Decreased Tumor Bioluminescence
Bioluminescent signal was tracked as a measure of tumor size throughout the study. Animals receiving the KD alone or in combination with HBO 2 T demonstrated a notable trend of slower tumor growth over time. This trend was more pronounced in KD+HBO 2 T mice and reflected the increase in survival time seen in these animals (Figures 1, 2). The difference in mean tumor size between KD+HBO 2 T and control animals at week 3 was statistically significant (p = 0.0062; Figure 2B). Day 21 ex vivo organ bioluminescence of KD+HBO 2 T mice demonstrated a trend of reduced metastatic tumors in animals compared to the SD group ( Figure 2). Spleen bioluminescence was significantly decreased in KD+HBO 2 T mice (p = 0.0266).

The KD Lowered Blood Glucose, Elevated Blood Ketones, and Decreased Body Weight
Prior to the study, initial blood glucose, ketone, and body weights were similar among the groups (data not shown). Blood glucose levels were lower in the KD-treated mice than in the SDtreated mice by day 7 (p,0.001; Figure 3). While all KD-fed mice demonstrated a trend of elevated blood ketone levels throughout the duration of the study, only the KD+HBO 2 T animals showed significantly increased ketones compared to controls on day 7 (p,0.001; Figure 3). By day 7, KD-fed mice lost approximately 10% of their initial body weight and maintained that weight for the duration of the study (Figure 4). Day 7 blood glucose and percent body weight change were significantly correlated to survival time (p = 0.0189 and p = 0.0001, respectively; Figure 5).

Discussion
Nearly a century after Otto Warburg reported the abnormal energy metabolism of cancer cells, renewed interest in the field has elucidated a plethora of novel therapeutic targets. Two promising treatments involve the use of HBO 2 T to reverse the cancerpromoting effects of tumor hypoxia and the use of the KD to limit the availability of glycolytic substrates to glucose-addicted cancer cells. Both therapies have been previously reported to possess anticancer effects [14,54,58,60]. Since these treatments are believed to work by targeting several overlapping mechanisms, we hypothesized that combining these non-toxic treatments would provide a powerful, synergistic anti-cancer effect. Furthermore, since metastasis is responsible for the overwhelming majority of cancer-related deaths, we tested the efficacy of these conjoined therapies on the VM-M3 mouse model of metastatic cancer [7,10]. We found that the KD fed ad libitum significantly increased mean survival time in mice with metastatic cancer (p = 0.0194; Figure 1). It is important to note that KD-fed animals lost approximately 10% of their body weight over the course of the study (Figure 4). It is well established that low carbohydrate, high fat ketogenic diets can cause body weight loss in overweight humans [21,22,61]. Ketogenic diets are also known to have an appetite suppressing effect which may contribute to body weight loss [20]. Along with appetite suppression, a potential contributing factor to the observed body weight loss is the possibility that mice found the KD to be less palatable and were self-restricting caloric intake. As calorie restriction is known to elicit profound anti-cancer effects, the ketogenic diet may inhibit cancer progression in part by indirect dietary energy restriction [6,38]. Fine and colleagues recently used a very low carbohydrate KD to promote stable disease or partial remission in patients with advanced metastatic cancer [62]. Fine's study demonstrated a correlation between blood ketones and response to the diet therapy, suggesting that ketone elevation itself also contributes to the anti-cancer efficacy of the KD.
As hypothesized, profound anti-cancer effects were observed in our metastatic mouse model after combining the KD with HBO 2 T. Combining these therapies nearly doubled survival time in mice with metastatic cancer, increasing mean survival time by  24 days compared to control animals (p = 0.0050; Figure 1). The KD+HBO 2 T-treated mice exhibited significantly decreased bioluminescence compared to controls at week 3 (p = 0.0062) and a trend of decreased tumor growth rate throughout the study ( Figure 2). By day 7, all animals on a ketogenic diet had significantly lower blood glucose levels than controls ( Figure 3). As it has been shown that tumor growth is directly correlated to blood glucose levels [63], this decrease in blood glucose concentration likely contributed to the trend of decreased tumor bioluminescence and rate of tumor growth seen in KD-fed animals ( Figure 2). Nebeling et al. demonstrated that the KD significantly decreased glucose uptake in pediatric brain tumor patients by FDG-PET analysis [64]. This clinical data suggests decreased glucose delivery to the tumor is a causal mechanism in KD treatment. All KD-fed mice showed a trend of elevated blood ketones throughout the study; however, only KD+HBO 2 T mice had significantly higher ketones than controls on day 7 ( Figure 3). As ketones are metabolized exclusively within the mitochondria, cancer cells with damaged mitochondria are unable to adequately use them for energy. Many cancers do not express the Succinyl-CoA: 3ketoacid CoA-Transferase (SCOT) enzyme which is required for ketone body metabolism [65,66]. In fact, bHB administration prevents healthy hippocampal neurons but not glioma cells from glucose withdrawal-induced cell death [24]. Furthermore, ketone bodies have anti-cancer effects themselves, possibly through inhibition of glycolytic enzymes [67]. Skinner and colleagues demonstrated that acetoacetate and bHB administration inhibits brain cancer cell viability in vitro [25]. Thus, the elevated ketone  A recent report by Listanti, et. al proposed that tumorassociated fibroblasts produce ketone bodies for cancer cells to use as fuel [68]. The authors have previously published several papers with similar findings [69,70,71]. In these studies, the authors created immortalized fibroblasts which were altered to overexpress rate-limiting enzymes in ketone body production, and co-cultured these cells with human breast cancer cells altered to overexpress enzymes involved in ketone body utilization. While this phenomenon may occur in the genetically altered culture system used by the authors, there is no evidence that this occurs naturally in cancer cells in vitro or in tumors in vivo. On the contrary, the literature as a whole strongly suggests that cancer cells cannot effectively use ketones for fuel. As described, most cancers do not express the SCOT enzyme which is necessary for ketone body utilization [65,66]. Several studies have reported a deficiency of cancer cells to metabolize ketone bodies in vitro [24,25]. Furthermore, it is widely accepted that ketone bodies are produced nearly exclusively from fatty acid b-oxidation in the liver. There is no known metabolic pathway by which fibroblasts can produce ketone bodies from glucose. Without additional compelling evidence to support these claims, we remain proponents of the notion that cancer cells cannot utilize ketone bodies as efficient energy substrates.
Potential concern may arise regarding the use of a diet therapy for cancer patients susceptible to cachexia. While low carbohydrate or ketogenic diets promote weight loss in overweight individuals, they are also known to spare muscle wasting during conditions of energy restriction and starvation [72,73,74,75]. In an animal model of cancer cachexia, administration of a low carbohydrate, high fat diet prevented weight loss of the animals while simultaneously decreasing tumor size [76]. Similar effects were described in human cancer patients [64,77]. The anticachexia effects of the KD are not surprising when considering a metabolic switch to fat metabolism and subsequent ketosis evolved as a method of sparing protein during prolonged fasting or starvation [72,78,79]. It makes sense that dietary-induced therapeutic ketosis in a cancer patient would prevent muscle wasting similarly as it does with athletes undergoing intense exercise [80]. Furthermore, when given as an adjuvant treatment to advanced cancer patients, the KD improves quality of life and enhances the efficacy of chemotherapy treatment in the clinic [81,82]. This and other emerging evidence calls into question the common medical advice of limiting fat consumption in overweight cancer patients [83].
Veech and colleagues described the mechanisms by which ketone metabolism protects cells from oxidative damage [74,78], while more recent evidence suggests that ketones function as HDAC inhibitors [84]. bHB metabolism results in an increased reduction of the NAD couple and increased oxidation of the coenzyme Q inside the mitochondria. Increased oxidation of Q decreases semiquinone levels, subsequently decreasing superoxide anion production [74]. Increased reduction of the NADP couple enhances regeneration of reduced glutathione, an important endogenous antioxidant [74]. Thus, ketone body metabolism protects cells from oxidative damage by decreasing ROS production and by enhancing endogenous antioxidant capabilities. As previously discussed, cancer cells are unable to effectively metabolize ketone bodies; therefore, we do not expect that ketones would confer the same protective effects onto the cancer cell. HBO 2 T increases ROS production within the cell which can lead to membrane lipid peroxidation and cell death [85]. Cancer cells with mitochondrial damage and chaotic perfusion naturally produce chronically elevated levels of ROS but are susceptible to oxidative damage-induced cell death with even modest increases in ROS [58,86]. We propose a potential mechanism of KD+HBO 2 T efficacy: the KD weakens cancer cells by glucose restriction and the inherent anti-cancer effects of ketone bodies while simultaneously conferring a protective advantage to the healthy tissue capable of ketone metabolism. This metabolic targeting sensitizes the cancer cells to HBO 2 T-induced ROS production and oxidative damage, contributing to the efficacy of combining KD with HBO 2 T. Additionally, ketone metabolism by the healthy tissues likely confers protection against the potential negative consequences of HBO 2 T (CNS oxygen toxicity) [87,88,89]. Recent in vivo studies support the neuroprotective effects of ketone esters [90,91]. These hypothetical mechanisms may contribute to the safety and efficacy of the KD+HBO 2 T combined therapy.
Stuhr and Moen recently published a comprehensive review of the literature on the use of HBO 2 T for cancer [59]. The authors concluded that HBO 2 T should be considered a safe treatment for patients with varying malignancies and that there is no convincing evidence its use promotes cancer progression or recurrence. In the literature, there are a substantial number of studies indicating that HBO 2 T can induce marked anti-cancer effects in vitro and in animal and human studies alike [58,59,92]. Evidence is mixed, however, as other studies reported no effect with HBO 2 T [58,59]. Indeed, in our present study, HBO 2 T alone did not improve the outcome of VM mice with metastatic cancer, but combining HBO 2 T with KD elicited a dramatic therapeutic effect. Perhaps adding the KD or another metabolic therapy (e.g. 2-deoxyglucose, 3-bromopyruvate) to HBO 2 T would produce similar results in these previously reported studies demonstrating no efficacy due to HBO 2 T alone. It is important to look for synergistic interactions between therapies which may increase the efficacy of cancer treatment. Scheck and coworkers reported complete remission without recurrence in 9 of 11 mice with glioma by combining the KD with radiation [93]. Marsh, et al. reported synergy between the restricted ketogenic diet and the glycolysis inhibitor 2deoxyglucose [94]. Might adding HBO 2 T to these combination therapies elicit even better results? Similarly, might the use of adjuvant therapies like KD and HBO 2 T enhance patient response to standard care?
Our study strongly suggests that combining a KD with HBO 2 T may be an effective non-toxic therapy for the treatment of metastatic cancer. The efficacy of combining these non-toxic treatments should be further studied to determine their potential for clinical use. Based on the reported evidence, it is highly likely that these therapies would not only contribute to cancer treatment on their own, but might also enhance the efficacy of current standard of care and improve the outcome of patients with metastatic disease.