Eric C. Woolf

The Ketogenic Diet Alters the Hypoxic Response and Affects Expression of Proteins Associated with Angiogenesis, Invasive Potential and Vascular Permeability in a Mouse Glioma Model

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.

Enhanced immunity in a mouse model of malignant glioma is mediated by a therapeutic ketogenic diet.

BackgroundGlioblastoma multiforme is a highly aggressive brain tumor with a poor prognosis, and advances in treatment have led to only marginal increases in overall survival. We and others have shown previously that the therapeutic ketogenic diet (KD) prolongs survival in mouse models of glioma, explained by both direct tumor growth inhibition and suppression of pro-inflammatory microenvironment conditions. The aim of this study is to assess the effects of the KD on the glioma reactive immune response.MethodsThe GL261-Luc2 intracranial mouse model of glioma was used to investigate the effects of the KD on the tumor-specific immune response. Tumor-infiltrating CD8+ T cells, CD4+ T cells and natural killer (NK) cells were analyzed by flow cytometry. The expression of immune inhibitory receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death 1 (PD-1) on CD8+ T cells were also analyzed by flow cytometry. Analysis of intracellular cytokine production was used to determine production of IFN, IL-2 and IFN- in tumor-infiltrating CD8+ T and natural killer (NK) cells and IL-10 production by T regulatory cells.ResultsWe demonstrate that mice fed the KD had increased tumor-reactive innate and adaptive immune responses, including increased cytokine production and cytolysis via tumor-reactive CD8+ T cells. Additionally, we saw that mice maintained on the KD had increased CD4 infiltration, while T regulatory cell numbers stayed consistent. Lastly, mice fed the KD had a significant reduction in immune inhibitory receptor expression as well as decreased inhibitory ligand expression on glioma cells.ConclusionsThe KD may work in part as an immune adjuvant, boosting tumor-reactive immune responses in the microenvironment by alleviating immune suppression. This evidence suggests that the KD increases tumor-reactive immune responses, and may have implications in combinational treatment approaches.

Tumor Metabolism, the Ketogenic Diet and β-Hydroxybutyrate: Novel Approaches to Adjuvant Brain Tumor Therapy

Malignant brain tumors are devastating despite aggressive treatments such as surgical resection, chemotherapy and radiation therapy. The average life expectancy of patients with newly diagnosed glioblastoma is approximately ~18 months. It is clear that increased survival of brain tumor patients requires the design of new therapeutic modalities, especially those that enhance currently available treatments and/or limit tumor growth. One novel therapeutic arena is the metabolic dysregulation that results in an increased need for glucose in tumor cells. This phenomenon suggests that a reduction in tumor growth could be achieved by decreasing glucose availability, which can be accomplished through pharmacological means or through the use of a high-fat, low-carbohydrate ketogenic diet (KD). The KD, as the name implies, also provides increased blood ketones to support the energy needs of normal tissues. Preclinical work from a number of laboratories has shown that the KD does indeed reduce tumor growth in vivo. In addition, the KD has been shown to reduce angiogenesis, inflammation, peri-tumoral edema, migration and invasion. Furthermore, this diet can enhance the activity of radiation and chemotherapy in a mouse model of glioma, thus increasing survival. Additional studies in vitro have indicated that increasing ketones such as β-hydroxybutyrate (βHB) in the absence of glucose reduction can also inhibit cell growth and potentiate the effects of chemotherapy and radiation. Thus, while we are only beginning to understand the pluripotent mechanisms through which the KD affects tumor growth and response to conventional therapies, the emerging data provide strong support for the use of a KD in the treatment of malignant gliomas. This has led to a limited number of clinical trials investigating the use of a KD in patients with primary and recurrent glioma.

Metabolism and glioma therapy

to provide energy and biomolecules regardless of the availability of oxygen. This shift toward increased glycolytic flux and away from the tricarboxylic acid cycle and oxidative phosphorylation occurs very early in tumorigenesis, prior to hypoxia while the tumor has sufficient oxygen. Since Warburg’s discovery, metabolism has been of interest in the cancer field, but it often seemed overshadowed by discoveries of oncogenes, tumor suppressor genes, growth factor pathways, molecular subtypes of cancers and so on. There is a resurgence of interest in metabolism as a central theme in cancer, and we continue to find that metabolic pathways intersect and often regulate key components of tumor initiation, progression and therapy response [6,7]. Thus, it has been suggested that one promising therapeutic strategy is to exploit the metabolic dysregulation seen in virtually all tumor cells. We now know that cancer metabolism is much more complex than just a higher rate of glycolysis. Mitochondrial bio genesis is also altered, and the cancer cell’s fate becomes reliant on the balance

Mathematical Analysis of Glioma Growth in a Murine Model

Five immunocompetent C57BL/6-cBrd/cBrd/Cr (albino C57BL/6) mice were injected with GL261-luc2 cells, a cell line sharing characteristics of human glioblastoma multiforme (GBM). The mice were imaged using magnetic resonance (MR) at five separate time points to characterize growth and development of the tumor. After 25 days, the final tumor volumes of the mice varied from 12 mm3 to 62 mm3, even though mice were inoculated from the same tumor cell line under carefully controlled conditions. We generated hypotheses to explore large variances in final tumor size and tested them with our simple reaction-diffusion model in both a 3-dimensional (3D) finite difference method and a 2-dimensional (2D) level set method. The parameters obtained from a best-fit procedure, designed to yield simulated tumors as close as possible to the observed ones, vary by an order of magnitude between the three mice analyzed in detail. These differences may reflect morphological and biological variability in tumor growth, as well as errors in the mathematical model, perhaps from an oversimplification of the tumor dynamics or nonidentifiability of parameters. Our results generate parameters that match other experimental in vitro and in vivo measurements. Additionally, we calculate wave speed, which matches with other rat and human measurements.

Probe-based three-dimensional confocal laser endomicroscopy of brain tumors: technical note

Background Confocal laser endomicroscopy (CLE) is used during fluorescence-guided brain tumor surgery for intraoperative microscopy of tumor tissue with cellular resolution. CLE could augment and expedite intraoperative decision-making and potentially aid in diagnosis and removal of tumor tissue. Objective To describe an extension of CLE imaging modality that produces Z-stack images and three-dimensional (3D) pseudocolored volumetric images. Materials and methods Hand-held probe-based CLE was used to collect images from GL261-luc2 gliomas in C57BL/6 mice and from human brain tumor biopsies. The mice were injected with fluorescein sodium (FNa) before imaging. Patients received FNa intraoperatively, and biopsies were imaged immediately in the operating room. Some specimens were counterstained with acridine orange, acriflavine, or Hoechst and imaged on a benchtop confocal microscope. CLE images at various depths were acquired automatically, compiled, rendered into 3D volumes using Fiji software and reviewed by a neuropathologist and neurosurgeons. Results CLE imaging, Z-stack acquisition, and 3D image rendering were performed using 19 mouse gliomas and 31 human tumors, including meningiomas, gliomas, and pituitary adenomas. Volumetric images and Z-stacks provided additional information about fluorescence signal distribution, cytoarchitecture, and the course of abnormal vasculature. Conclusion 3D and Z-stack CLE imaging is a unique new option for live intraoperative endomicroscopy of brain tumors. The 3D images afford an increased spatial understanding of tumor cellular architecture and visualization of related structures compared with two-dimensional images. Future application of specific fluorescent probes could benefit from this rapid in vivo imaging technology for interrogation of brain tumor tissue.

The therapeutic ketogenic diet as an adjuvant therapy for cancer

T T cell receptor (TCR) repertoire holds clues to the functioning of the immune system. Profiling the repertoire can provide a systemic view of the health of an individual. T-seq, our novel method of sequencing the T cell receptor (TCR) repertoire, enables its unbiased sampling. This has enabled inexpensive, deep profiling of the TCR repertoire in both mouse and human T cells. The method does not require T cell isolation and can be used to study infiltrates in tissues. By applying it to a variety of samples from mouse and human, we have improved the annotation of the TCR loci in both genomes. We have also characterized the RSS signals and identified evolutionary constraints, as well as their potential to shape the statistics of segment usage. We describe methods of characterizing the usage of V and J segments in the alpha and beta chains and comparing TCR repertoire data across samples. This study enhances our understanding of the “normal” TCR repertoire, and provides the tools and annotations needed to characterize disease states, in order to help identify biomarkers and potential targets of therapy. We propose methods of identifying signatures of repertoires in cancer and the potential implications of this signature for outcomes of cancer immunotherapy. We are using these tools to study the role of the TCR repertoire in disease progression and potential for use of the diversity of the TCR repertoire as a prognostic indicator.

Abstract B73: Ketogenic diet enhances immunity to glioblastoma

Glioblastomas are the most common and malignant brain tumor in adults and account for over 50% of all gliomas. Current standard of care combines surgical resection with temozolomide chemotherapy and radiotherapy, but 5 year survival rates remain less than 20%. There is a critical need for new therapies that increase efficacy without increasing toxicity. One therapeutic target can be found in the aberrant metabolism that is a hallmark of cancer. We have previously used an intracranial syngeneic immunocompetent mouse model of malignant glioma to demonstrate that targeting metabolism using a ketogenic diet reduced tumor burden and increased survival. The ketogenic diet is a high fat, low carbohydrate plus protein diet used for the treatment of refractory pediatric epilepsy. Mice maintained on a ketogenic diet (KetoCal®, Nutricia North America, Gaithersburg, MD) had a reduction in inflammation and peri-tumoral edema, prompting us to question the effects of the ketogenic diet on the infiltration and the functionality of glioblastoma-reactive immune cells. Immune cells were isolated from malignant gliomas from mice treated with either the ketogenic diet or a standard diet. Glioblastoma-specific immune cells were tested for functionality via cytokine production, capacity for cytotoxicity, and amount of infiltration. We found the ketogenic diet significantly decreased expression of the inhibitory receptors CTLA4 and PD-1 on tumor-infiltrating T cells, and coincided with a reduction in inhibitory ligand expression on the tumor. Moreover, this reduced T cell tolerance at the tumor site allowed CD8+ T cells to produce IFNγ and IL-2, and regain cytotoxic functions. Additionally, the ketogenic diet caused a reduction in immunosuppressive cytokine production via regulatory T cells. Innate immune responses to the tumor were also altered in mice maintained on the ketogenic diet, as natural killer cells infiltrating the tumor were able to produce more IFNγ and TNF in response to GL261-luc2 cells in vitro. Overall, the ketogenic diet may be an attractive adjuvant therapy to overcome several immune escape mechanisms in gliomas by decreasing regulatory T cell suppression of effector T cells and increasing CD8+ T cell killing and cytokine production, ultimately leading to increased tumor immune mediated rejection. Citation Format: John L. Johnson, Eric Woolf, Danielle M. Lussier, Ken S. Brooks, Joseph N. Blattman, Adrienne C. Scheck. Ketogenic diet enhances immunity to glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B73.