A century ago, Otto Warburg discovered that cancer cells display a unique metabolic phenotype of lactate fermentation in the presence of oxygen. This phenotype, known as the Warburg effect, enables tumor visualization using fluorodeoxyglucose positron emission tomography (FDG‐PET) scans owing to the elevated rate of glucose consumption in most cancers. Metabolic therapies can exploit this phenotype, offering novel therapeutic directions aside from the classically targeted cytotoxic and gene‐based therapies. The Warburg effect exposes a fundamental weakness of cancer cells, reliance on excess glucose for survival and maximal proliferation. Fasting, calorie restriction (CR) and the carbohydrate‐restricted ketogenic diet have been successfully used to limit glucose availability and slow cancer progression in a variety of animal models and human studies.1-9 These dietary manipulations produce a physiological metabolic shift to an unfavorable environment for glucose‐dependent cancer cells. Previously, the anticancer effects of these dietary manipulations have largely been attributed to decreased circulating blood glucose, which limits energy substrates for cancer cells. New evidence suggests, however, that the physiological state of ketosis and elevated circulating ketones also have anticancer effects.

Recently, Fine et al. demonstrated that a carbohydrate‐restricted ketogenic diet inhibited disease progression and promoted partial remission in patients with advanced metastatic cancers from various tissue origins.10 On average, the patients did not exhibit a drop in glucose from baseline, suggesting that decreased glucose availability was not the sole or primary cause of efficacy. Interestingly, the study found that the most important factor dictating the patients' response to therapy was the degree of elevated ketosis from baseline. Indeed, a prominent metabolic shift to higher levels of ketosis correlated with reduced disease progression, stable disease or partial regression. A small number of reports have investigated the effects of ketones on cancer growth in vitro. Acetoacetate (AcAc) and β‐hydroxybutyrate (βHB) are the two most abundant and physiologically relevant of the three ketone bodies. Acetone is produced as a nonenzymatic byproduct of AcAc metabolism and is rapidly excreted in the lungs. AcAc supplementation inhibited proliferation and ATP production in seven aggressive human colon and breast cancer cell lines but did not affect proliferation in healthy primary fibroblasts.11 Similarly, βHB inhibited proliferation of transformed lymphoblasts, HeLa cells and melanoma cells in a dose‐dependent manner up to 20 mM.6 In neuroblastoma cells, βHB and AcAc supplementation decreased viability and increased apoptosis, but had no effect on fibroblasts.12

Although ketone bodies are efficient energy substrates for healthy extrahepatic tissues, 13 cancer cells cannot effectively use them for energy. Widespread mitochondrial pathology has been observed in most if not all tumors examined, including decreased mitochondrial number, abnormal ultrastructural morphology, mitochondrial swelling, abnormal fusion–fission, partial or total cristolysis, mtDNA mutations, altered mitochondrial membrane potential and abnormal mitochondrial enzyme presence or function, among others.14-19 These defects in mitochondrial structure and function impair respiratory capacity and force a reliance on substrate‐level phosphorylation for survival.20 As ketone bodies are metabolized exclusively within the mitochondria, cancer cells with impaired mitochondrial function are unable to efficiently metabolize ketone bodies for energy. Indeed, unlike healthy cells, ketone bodies fail to rescue glioma cells from glucose withdrawal‐induced death.21

Although mitochondrial dysfunction explains the inability of cancer cells to effectively use ketones for energy, the anticancer effects of ketones in a normal glucose in vitro environment are not immediately clear. Upon closer examination, ketone bodies possess many characteristics that can impair cancer cell survival and proliferation. (i) Ketone bodies inhibit glycolysis, thus decreasing the main pathway of energy production for cancer cells.22 (ii) Cancer cells thrive in an environment of elevated reactive oxygen species (ROS) production but are very sensitive to even small changes in redox status.23 Ketones decrease mitochondrial ROS production and enhance endogenous antioxidant defenses in normal cells, but not in cancer cells.13 Ketone metabolism in healthy cells near the tumor may inhibit cancer cell growth by creating a less favorable redox environment for their survival. (iii) Ketone bodies are transported into the cell through the monocarboxylate transporters (MCTs), which are also responsible for lactate export. It has been shown that inhibiting MCT1 activity or inhibiting lactate export from the cell dramatically decreases cancer cell growth and survival.24 Ketones may impair cancer cells indirectly by competitive inhibition of the MCTs, decreasing critical lactate export from the cell. (iv) Recently, Verdin and coworkers demonstrated that βHB acts as an endogenous HDAC inhibitor at millimolar concentrations easily achieved through fasting, CR or ketone supplementation such as with a ketone ester (KE).25 Thus, ketone bodies may elicit their anticancer effects by altering the expression of oncogenes and tumor suppressor genes under control of the cancer epigenome.26 Clearly, ketone bodies exhibit several unique characteristics that support their use as a metabolic therapy for cancer.

The Warburg effect is especially prevalent in aggressive cancers and metastatic cells. Metastasis, the spreading of a primary tumor to distal locations, is the primary cause of cancer morbidity and mortality and is responsible for more than 90% of cancer‐related deaths.27 To make significant strides toward long‐term cancer management, we must evaluate therapies that are effective against late‐stage metastatic cancers. The major obstacle preventing the discovery of such treatments has been the lack of animal models that mimic the true metastatic phenotype and accurately predict the clinical success of therapeutics. The VM‐M3 model of metastatic cancer is a novel murine model that closely mimics the natural progression of cancer invasion and metastasis.28 The original VM‐M3 tumor arose as a spontaneous brain tumor in a VM/Dk inbred mouse. The VM‐M3 tumor was adapted to cell culture and transduced with the firefly luciferase gene to allow for in vivo bioluminescent tracking of tumor growth.29 VM‐M3 cells express many in vitro and in vivo characteristics of human glioblastoma multiforme with macrophage or microglial properties.28-30 When implanted subcutaneously, the VM‐M3 cells are morphologically similar to histiocytes and rapidly produce systemic metastasis, forming tumors in all major organ systems, most notably the liver, lung, kidney, spleen, brain and bone.28 The VM‐M3 model of metastatic cancer spreads naturally in an immunocompetent host, and chemotherapeutic agents inhibit metastatic spread in this model similar to their observed effects in humans.31 The VM‐M3 model has many advantages that support its use as a representation of the true metastatic disease state and has therefore been used in our study.

Although the literature strongly suggests that ketone bodies impede cancer growth in vitro, the in vivo efficacy of dietary ketone supplementation has not been adequately examined. It is possible to raise blood ketone levels without the need for carbohydrate restriction by administering a source of supplemental ketones or ketone precursors. 1,3‐Butanediol (BD) is a commercially available food additive and hypoglycemic agent that is converted to βHB by the liver.32, 33 The KE elevates both AcAc and βHB in a dose‐dependent manner to levels beyond what can be achieved with the KD or therapeutic fasting.34 Oral administrations of BD and KE have been shown to elevate blood ketones for at least 240 min in rats.34 As ketone bodies appear to elicit anticancer effects, and metastasis is the most significant obstacle in the successful treatment of neoplasms, we tested the efficacy of ketone supplementation in the VM‐M3 cell line and mouse model of metastatic cancer.