Mark C. Preul

INTRODUCTION: The ketogenic diet (KD) is a high-fat, low-carbohydrate diet that alters metabolism by increasing the level of ketone bodies in the blood. KetoCal® (KC) is a nutritionally complete, commercially available 4:1 (fat:carbohydrate+protein) ketogenic formula that is an effective non-pharmacologic treatment for the management of refractory pediatric epilepsy. Diet-induced ketosis causes changes to brain homeostasis that have potential for the treatment of other neurological diseases such as malignant gliomas. METHODS: We used an intracranial bioluminescent mouse model of malignant glioma. Following implantation animals were maintained on standard diet (SD) or KC. The mice received 2×4 Gy of whole brain radiation and tumor growth was followed by in vivo imaging. RESULTS: Animals fed KC had elevated levels of β-hydroxybutyrate (p = 0.0173) and an increased median survival of approximately 5 days relative to animals maintained on SD. KC plus radiation treatment were more than additive, and in 9 of 11 irradiated animals maintained on KC the bioluminescent signal from the tumor cells diminished below the level of detection (p<0.0001). Animals were switched to SD 101 days after implantation and no signs of tumor recurrence were seen for over 200 days. CONCLUSIONS: KC significantly enhances the anti-tumor effect of radiation. This suggests that cellular metabolic alterations induced through KC may be useful as an adjuvant to the current standard of care for the treatment of human malignant gliomas.

BACKGROUND: Malignant brain tumors affect people of all ages and are the second leading cause of cancer deaths in children. While current treatments are effective and improve survival, there remains a substantial need for more efficacious therapeutic modalities. The ketogenic diet (KD) - a high-fat, low-carbohydrate treatment for medically refractory epilepsy - has been suggested as an alternative strategy to inhibit tumor growth by altering intrinsic metabolism, especially by inducing glycopenia. METHODS: Here, we examined the effects of an experimental KD on a mouse model of glioma, and compared patterns of gene expression in tumors vs. normal brain from animals fed either a KD or a standard diet. RESULTS: Animals received intracranial injections of bioluminescent GL261-luc cells and tumor growth was followed in vivo. KD treatment significantly reduced the rate of tumor growth and prolonged survival. Further, the KD reduced reactive oxygen species (ROS) production in tumor cells. Gene expression profiling demonstrated that the KD induces an overall reversion to expression patterns seen in non-tumor specimens. Notably, genes involved in modulating ROS levels and oxidative stress were altered, including those encoding cyclooxygenase 2, glutathione peroxidases 3 and 7, and periredoxin 4. CONCLUSIONS: Our data demonstrate that the KD improves survivability in our mouse model of glioma, and suggests that the mechanisms accounting for this protective effect likely involve complex alterations in cellular metabolism beyond simply a reduction in glucose.

BACKGROUND: The successful treatment of malignant gliomas remains a challenge despite the current standard of care, which consists of surgery, radiation and temozolomide. Advances in the survival of brain cancer patients require the design of new therapeutic approaches that take advantage of common phenotypes such as the altered metabolism found in cancer cells. It has therefore been postulated that the high-fat, low-carbohydrate, adequate protein ketogenic diet (KD) may be useful in the treatment of brain tumors. We have demonstrated that the KD enhances survival and potentiates standard therapy in a mouse model of malignant glioma, yet the mechanisms are not fully understood. METHODS: To explore the effects of the KD on various aspects of tumor growth and progression, we used the immunocompetent, syngeneic GL261-Luc2 mouse model of malignant glioma. RESULTS: Tumors from animals maintained on KD showed reduced expression of the hypoxia marker carbonic anhydrase 9, hypoxia inducible factor 1-alpha, and decreased activation of nuclear factor kappa B. Additionally, tumors from animals maintained on KD had reduced tumor microvasculature and decreased expression of vascular endothelial growth factor receptor 2, matrix metalloproteinase-2 and vimentin. Peritumoral edema was significantly reduced in animals fed the KD and protein analyses showed altered expression of zona occludens-1 and aquaporin-4. CONCLUSIONS: The KD directly or indirectly alters the expression of several proteins involved in malignant progression and may be a useful tool for the treatment of gliomas.

OBJECT: Infiltrative tumor resection is based on regional (macroscopic) imaging identification of tumorous tissue and the attempt to delineate invasive tumor margins in macroscopically normal-appearing tissue, while preserving normal brain tissue. The authors tested miniaturized confocal fiberoptic endomicroscopy by using a near-infrared (NIR) imaging system with indocyanine green (ICG) as an in vivo tool to identify infiltrating glioblastoma cells and tumor margins. METHODS: Thirty mice underwent craniectomy and imaging in vivo 14 days after implantation with GL261-luc cells. A 0.4 mg/kg injection of ICG was administered intravenously. The NIR images of normal brain, obvious tumor, and peritumoral zones were collected using the handheld confocal endomicroscope probe. Histological samples were acquired from matching imaged areas for correlation of tissue images. RESULTS: In vivo NIR wavelength confocal endomicroscopy with ICG detects fluorescence of tumor cells. The NIR and ICG macroscopic imaging performed using a surgical microscope correlated generally to tumor and peritumor regions, but NIR confocal endomicroscopy performed using ICG revealed individual tumor cells and satellites within peritumoral tissue; a definitive tumor border; and striking fluorescent microvascular, cellular, and subcellular structures (for example, mitoses, nuclei) in various tumor regions correlating with standard clinical histological features and known tissue architecture. CONCLUSIONS: Macroscopic fluorescence was effective for gross tumor detection, but NIR confocal endomicroscopy performed using ICG enhanced sensitivity of tumor detection, providing real-time true microscopic histological information precisely related to the site of imaging. This first-time use of such NIR technology to detect cancer suggests that combined macroscopic and microscopic in vivo ICG imaging could allow interactive identification of microscopic tumor cell infiltration into the brain, substantially improving intraoperative decisions.

INTRODUCTION: Recent developments in optical science and image processing have miniaturized the components required for confocal microscopy. Clinical confocal imaging applications have emerged, including assessment of colonic mucosal dysplasia during colonoscopy. We present our initial experience with handheld, miniaturized confocal imaging in a murine brain tumor model. METHODS: Twelve C57/BL6 mice were implanted intracranially with 10(5) GL261 glioblastoma cells. The brains of 6 anesthetized mice each at 14 and 21 days after implantation were exposed surgically, and the brain surface was imaged using a handheld confocal probe affixed to a stereotactic frame. The probe was moved systematically over regions of normal and tumor-containing tissue. Intravenous fluorescein and topical acriflavine contrast agents were used. Biopsies were obtained at each imaging site beneath the probe and assessed histologically. Mice were killed after imaging. RESULTS: Handheld confocal imaging produced exquisite images, well-correlated with corresponding histologic sections, of cellular shape and tissue architecture in murine brain infiltrated by glial neoplasm. Reproducible patterns of cortical vasculature, as well as normal gray and white matter, were identified. Imaging effectively distinguished between tumor and nontumor tissue, including infiltrative tumor margins. Margins were easily identified by observers without prior neuropathology training after minimum experience with the technology. CONCLUSION: Miniaturized handheld confocal imaging may assist neurosurgeons in detecting infiltrative brain tumor margins during surgery. It may help to avoid sampling error during biopsy of heterogeneous glial neoplasms, with the potential to supplement conventional intraoperative frozen section pathology. Clinical trials are warranted on the basis of these promising initial results.