A Supplemented High-Fat Low-CarbohydrateDiet for the Treatment of GlioblastomaRegina T. Martuscello1,2, Vinata Vedam-Mai1,3, David J. McCarthy1, Michael E. Schmoll1,Musa A. Jundi1, Christopher D. Louviere1, Benjamin G. Griffith1, Colby L. Skinner1,Oleg Suslov1, Loic P. Deleyrolle1, and Brent A. Reynolds1,2
Abstract
Purpose:Dysregulated energetics coupled with uncontrolledproliferation has become a hallmark of cancer, leading toincreased interest in metabolic therapies. Glioblastoma (GB)is highly malignant, very metabolically active, and typicallyresistant to current therapies. Dietary treatment options basedon glucose deprivation have been explored using a restrictiveketogenic diet (KD), with positive anticancer reports. However,negative side effects and a lack of palatability make the KDdifficult to implement in an adult population. Hence, wedeveloped a less stringent, supplemented high-fat low-carbo-hydrate (sHFLC) diet that mimics the metabolic and antitumoreffects of the KD, maintains a stable nutritional profile, andpresents an alternative clinical option for diverse patientpopulations.
Experimental Design: The dietary paradigm was tested in vitroand in vivo, utilizing multiple patient-derived gliomasphere lines.
Cellular proliferation, clonogenic frequency, and tumor stem cellpopulation effects were determined in vitro using the neurosphereassay (NSA). Antitumor efficacy was tested in vivo in preclinicalxenograft models and mechanistic regulation via the mTORpathway was explored.
Results:Reducing glucose in vitro to physiologic levels, coupledwith ketone supplementation, inhibits proliferation of GB cellsand reduces tumor stem cell expansion. In vivo, whilemaintaininganimal health, the sHFLC diet significantly reduces the growth oftumor cells in a subcutaneous model of tumor progression andincreases survival in an orthotopic xenograft model. Dietary-mediated anticancer effects correlate with the reduction of mTOReffector expression.
Conclusions: We demonstrate that the sHFLC diet is a viabletreatment alternative to the KD, and should be considered forclinical testing. Clin Cancer Res; 22(10); 2482–95. �2015 AACR.
IntroductionGlioblastoma (GB) is the most common high-grade glioma in
adults, with extremely poor prognosis. Tentacle-like projectionsand pseudopalisading necrosis integrate into normal brain tissuemaking complete surgical resection difficult. Extreme cellularheterogeneity, extensive genetic aberrations, and inadequate earlydetection make effective long-term control of GB challenging.Current standard of care (SOC) is limited to surgery, radiation,and chemotherapy (temozolomide/TMZ), with median survivalof 9 to 12 months and 5-year survival less than 5% (1).
Glucose metabolism and the Warburg Effect have gainedtraction as a potential tumor weakness and exploitable treatmentarea. Where normal cells utilize glucose for high-yield energyproduction in the mitochondria (1:36ATP), tumor cells demandhigher levels of glucose for diminished energy production, vialactate in the cytosol (1:4ATP) and nucleotide synthesis in thepentose phosphate pathway. This metabolic characteristic,termed the Warburg Effect, is an essential byproduct of rapidcellular proliferation and promoted during tumorigenesis byoncogenic metabolic reprogramming. Hence tumor cells acquirethe ability to sustain proliferative signaling mechanisms, whichsubsequently promotes malignant glycolysis (2). The PI3K/Akt/mTOR pathway plays a central role in human cancers by way ofconstitutive activity, increased growth factor expression and gly-colytic activation, resulting in feed-forward loops of glucoseconsumption, proliferation and survival (3). PTEN, a key regu-lator of the PI3K/Akt/mTOR pathway, is frequently inactivated inGB.
Research into dysregulated cellular metabolism has given riseto the notion that dietary therapies for cancer patients may havesignificant clinical utility. GBhas beenproposed tobe apromisingcandidate for dietary intervention due to its substantial relianceand utilization of glucose (4, 5). At the forefront of dietaryanticancer therapy is the ketogenic diet (KD), which is a high-fat, low-carbohydrate, low-protein diet, used for decades to treatrefractory epileptic seizures. Extreme carbohydrate restrictionmimics a fasting state, resulting in reduction of blood glucoseand induction of ketone bodies (e.g., b-hydroxybutyrate/BHB;
1Department of Neurosurgery, College of Medicine, University of Flor-ida, Gainesville, Florida. 2Interdisciplinary Program in BiomedicalSciences, Neuroscience, College of Medicine, University of Florida,Gainesville, Florida. 3Center for Movement Disorders and Neuro-res-toration, University of Florida, Gainesville, Florida.
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
Corresponding Authors: Brent A. Reynolds, Department of Neurosurgery,McKnight Brain Institute, 1149 S. Newell Drive, L2-100, Gainesville, FL 32611.Phone: 352-273-8476; Fax: 352-392-8413; E-mail: [email protected]; LoicP. Deleyrolle, Department of Neurosurgery, McKnight Brain Institute, 1149 S.Newell Drive, L2-100, Gainesville, FL 32611. Phone: 352-273-8583; Fax: 352-392-8413; E-mail: [email protected]
ref. 6). Ketone bodies are suitable energy replacements for normalcellswith functionalmitochondria (7), but have been shown tobeunsuitable for tumor cells (8,9), as tumor cell mitochondrialfunctions are dysregulated (10). Existing preclinical data supportthe KD (11–13) and a calorie-restricted KD (RKD; refs. 14, 15) inthe treatment of brain cancer by diminishing tumor growth andincreasing animal survival. Clinical reports (5), case reports(16,17), and pilot trials (18–20) have demonstrated that the KDis safe, has low toxicity, and is applicable to cancer patients.
These publications note that there are obstacles in its use as acancer treatment due to undesirability for long-term use, withsome reporting mild-to-severe side effects (21). It has beenproposed that more endurable dietary regimens should be devel-oped and tested, as not all patients may tolerate the same dietaryrestrictions (22). Therefore, we sought to develop a less restrictiveKD-like diet that would exhibit the same physiologic phenotypeand antitumor efficacy. By supplementing a high-fat, low-carbo-hydrate (sHFLC), moderate protein diet with specialized medi-um-chain triglycerides [MCT; 60%(30%):30%:10%::Fat(MCT):Protein:Carb], we hypothesize that a more balanced diet can beimplemented, resulting in diminished tumor progression. MCTswere specifically chosen based on carbon chain lengths (C8:C10::97%:3%), which allow them to rapidly diffuse from thegastrointestinal tract into the hepatic portal system and traveldirectly to the liver where they are converted into ketone bodies(6). We believe it is possible to provide a more nutritionallycomplete, flexible, and palatable anticancer diet with the sHFLC,which could target a diverse patient population and increasepatient compliance.
Materials and Methods1
CellsPatient glioblastoma tumors were dissociated and cultured as
previously published (23). All lines tested and authenticatedvia STR-profiling following ICLAC protocols. Culture mediaare serum free, supplemented with EGF at 20 ng/mL. Glucose
restrictedmedia were tested with a glucometer and supplementedwith glucose at plating and every-other-day for the 7-day passagecycle. BHB was administered in themedia at plating day only andmeasured at day 1 andpassaging day 7using theOneTouchmeter.
ImagesAfter sphere formation, cells were fixed with PFA, stained with
DAPI, and imaged using Leica DM 2000 2.5� fluorescent micro-scope. Images were quantified using Macnification.
AnimalsAll experiments were approved by IACUC.NOD/SCID animals
were housed under standard animal husbandry procedures.Transplants: Animals were transplanted per previously publishedprotocols (24). For subcutaneous xenografts, dissociated glioma-spheres were transplanted (1�106). Endpoint criteria of 15�15mm (1766mm3) were used. For intracranial xenografts, animalswere stereotactically injected with dissociated gliomaspheres(2�105) into the striatum (2 ML, and 2.5 DV). Endpoint criteriawere >20% body weight loss, BCS �2, and neurologic deficits.
Diet creation and deliveryThe ketogenic diet was readymade Bioserv #F3666. The sHFLC
and control diets were created using ingredients from Purina TestDiet. The sHFLC diet final caloric% was 10:30:30(30) carbohy-drates:protein:fat(MCTs; 3.2:2.8:1 fats:protein:carb). The sHFLCdiet provided 5.66 kcal/g gross energy where fat, carbohydrates,protein, and fiber comprised 404 g/kg, 125 g/kg, 355 g/kg, 73.12g/kg, respectively. The control diet caloric% was 55:20:25 carbo-hydrates:protein:fat (1:1.8:4.9 fats:protein:carb). The control dietdelivered 4.67 kcal/g gross energy where fat, carbohydrates, pro-tein, and fiber comprised 115 g/kg, 570 g/kg, 208 g/kg, 72 g/kg,respectively. Food (ad libitum)was changed daily. Upon SC tumorpalpation, (1�1mm) animals were randomized and assignedrespective diets. Intracranial animalswere randomized post-trans-plant and assigned respective diets 5 days after final transplant.
Tumor occupancyQuantification of tumor occupancy was determined using
ImageJ. Three images for each brain (4 control, 3 sHFLC, 3 KD)were taken from rostral, medial, and caudal positions of thetumor.
IHCTissue was formaldehyde fixed, cryopreserved, or embedded in
paraffin. Sections were blocked and incubated in primary anti-body overnight at 4�C. Fluorescent secondary antibody wasapplied for 1 hour at RT. Slides were coverslipped-using Vecta-Shield with DAPI. Biotinylated secondary antibody was appliedfor 2 hours at RT followed by 1 hour Vectastain Elite ABC kit andDAB peroxidase kit. Slides were coverslipped with Cytoseal.
Western blot analysisProteins were extracted from subcutaneous tumors and quan-
tified using Qubit. Equal amounts of protein were loaded intoeach well (20–40 mg). Gels were run for 30 minutes at 200 V andthen transferred using a xCell-II blot module (1 hour, 35A, RT).Proteins of interest were detected using chemoluminescense.Membranes were exposed (Kodak), stripped, and probed foractin.1For detailed experimental procedures please see supplemental methods file.
Translational Relevance
The emerging role of metabolism in many types of cancersprovides a novel target for new therapeutics. A possibility isdietary therapies that are designed to take advantage of theunique energetic needs of tumor cells. One such option is theuse of a very high-fat and low-carbohydrate diet (90%:5%,respectively) called theKetogenicDiet (KD). This diet has beenused to treat epilepsy for nearly 90 years, and recently itsapplication as a cancer therapeutic has been explored.Although preclinical studies demonstrate its efficacy and clin-ical case reports support its feasibility, the KD is difficult toimplement due to its stringent nature. Here, we report thedevelopment of an sHFLC, which mimics the main physio-logic effects of the KD, namely reduced glucose and increasedketones. In addition, the sHFLC diet is able to reduce glio-blastoma (GB) tumor cell proliferation and extend lifespan inGB animal models.
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Flow cytometryCells from culture and ex-vivo cells from SC tumors were run on
a BD Biosciences LSRII flow cytometer. Briefly, 0.5�106 cells wereblocked, permeabilized, and incubated with primary antibodiesovernight at 4�C. Secondary antibody was applied for 1 hour atRT. Flow analysis was performed using FlowJo software.
RNA isolation and RT2 PCR Human mTOR ProfilerTotal RNAwas isolated fromSC tumors usingRNeasy PlusMini
RNA isolation kit. RT2first strands andRT2 SYBRGreenROXqPCRMastermix were utilized. RT-PCR was performed using Qiagen(PAHS-098ZE-4), on ABI 7900HT. Housekeeping genes weredetermined using NormFinder.
ResultsRestricted glucose slows proliferation of patient-derivedgliomaspheres
Using four different previously published GB patient-derivedcell lines (L0, L1, L2, and S3; refs. 24–26), we examined the effectsof glucose restriction in-vitro. For this publication, L0will be usedas the primary cell line due to its robust proliferative activity, andL2 will be used as the secondary line since it closely resembles theproliferative and metabolic activity of L0. Cells were grownunder three glucose conditions: normal (glucose) media (NG:500þmg/dL), physiologic (glucose) media (PG: 90–110mg/dL),and physiologic-low (glucose) media (LG: 65–80 mg/dL). Figure1A shows altering glucose concentrations in the media signifi-cantly reduced L0 cell proliferation over time. The cell linesrevealed a significant reduction in mean expansion under PGand LGwhen compared withNG (Supplementary Fig. S1A–S1C),as well as a significant difference between PG and LG media (Fig.1B), as confirmed by a MTT Assay (Fig. 1C). To assess thereduction in fold expansion, L0 was analyzed for proliferation(Ki-67-Fig. 1D; MCM2-Fig. 1E) and apoptosis markers (active-caspase3-Fig. 1F). Lowering glucose concentrations resulted in asignificant reduction in Ki-67 and MCM2 expression, in both PGandLG, aswell as a significant increase in active caspase-3betweenNG and LG. Therefore, alterations in glucose availability, to levelsequivalent to a low glucose state, are sufficient to slow theproliferation of gliomaspheres while concomitantly increasingapoptosis.
The clonogenic frequency and proliferative rate of individualclones (24) were assessed in 96-well culture plates under allconditions (Fig. 1G). Therewas a statistically significant reductionin both the clonogenic frequency (Fig. 1H) and the mean prolif-erative rate of each clone (Fig. 1I), in all lines (Supplementary Fig.S1D—S1I). Quantification of the self-renewing stem cell sym-metrical division rate (KLL; ref. 24) demonstrated a significantdecrease in cancer stem cell expansion under reduced glucoseconditions (Fig. 1J). The combination of diminished stem celldivision rates, cellular fold expansion, and proliferation markersindicates that lowering glucose affects not only the putative stemcell population, but also the non-stem cell population.
Ketone treatment in vitro further suppresses proliferation ofpatient-derived GB cells
To determine ketone effects in vitro and model the sHFLCdietary phenotype (low glucose and high ketones), a doseresponse experiment was performed. BHB treatment in allglucose conditions resulted in a dose-dependent reduction in
fold expansion beginning at 4 mmol/L (Fig. 2A). BHB EC50s werecalculated for each glucose condition (Fig. 2A).On the basis of theBHB EC50 of L0 from PG, as well as the knowledge that this is anachievable, efficacious ketosis plasma concentration for patientson a high-fat diet (7), all in vitro experiments were performedwith4 mmol/L BHB (KTX). The MTT assay also showed reduction inenzymatic activity post BHB treatment beginning at 1mmol/L, butwithout dose dependence (Supplementary Fig. S2A—S2C). Toconfirm the reduction in fold expansion, we performed 50 con-secutive single passages with 3-glucose concentrations with KTX,and we still observed a significant reduction in fold expansion(Fig. 2B) and MTT enzyme activity (Fig. 2C) in all lines (Supple-mentary Fig. S2D–S2E). We assessed proliferation and apoptosisin L0 KTX and found an enhanced reduction in Ki-67 (Fig. 2D)andMCM2 (Fig. 2E) expression, but no increase in active caspase-3 activity (Fig. 2F) post KTX. Clonogenic frequency, proliferationrate of individual clones, and tumor stem cell division rate wereeach determined under restricted glucose, and in conjunctionwith KTX (Fig. 2G). A significant reduction was observed inclonogenic frequency (Fig. 2H) and mean proliferation rate ofeach clone (Fig. 2I) in each glucose media when exposed to KTX,in all lines (Supplementary Fig. S2G–S2L). KLL quantificationdemonstrated a significant reduction in stem cell division rate inall KTX groups compared with glucose controls, with a significantreduction in the low glucose and KTX combination (Fig. 2J) in alllines (Supplementary Fig. S2H, S2J, and S2L]. Together, thisevidence supports the potential anti-tumorigenic effect of thesHFLC dietary phenotype by diminishing the proliferative poten-tial of the putative stem cell and non-stem cell populations.
The sHFLC diet is safe and nutritionally completeDietary breakdown for all xenograft experiments is shown in
Supplementary Fig. Table 1. Detailed descriptions of diet creationand dietary timelines are provided in the Supplemental Methods.Animals placed on the sHFLC and KD had a significant reductionin blood glucose, above hypoglycemic levels, compared to con-trols, with no difference between the sHFLC and KD (Fig. 3A andSupplementary Fig. S3A). Blood ketones were significantlyincreased in mice maintained on the sHFLC and KD to a safelevel, with a statistical difference between the groups (Fig. 3B andSupplementary Fig. S3B). To compare blood glucose and ketonelevels among different anticancer dietary therapies, a simpleglucose ketone index (GKI) is used. The GKI is a single number,and can be used both clinically and preclinically, to identify atherapeutic zone (27). On the basis of this formulation, ouraverage calculated GKIs are 24.4 � 7.14, 3.1 � 1.07, and 1.94� 0.67 for the control, sHFLC and KD, respectively. These valuesare comparable with other published preclinical studies reportinga therapeutic effect using a RKD (range of 1.8–5.7; ref. 27). Tocompare nutritional differences and health status of mice onsHFLC and KD, body weight and blood analyses were performed.Patients on RKD report losing approximately 20% of their bodyweight (28), whereas patients on KD report initial body weightfat-loss followed by stabilization (29). Surprisingly, subcutane-ous-L0 mice fed the sHFLC diet had a significant increase in bodyweight compared with the KD and control (Fig. 3C). In otherxenograft experiments, sHFLC-fed mice had a significant increasein bodyweight comparedwith KD-fedmice only (SupplementaryFig. S3C—S3E]. This may be due to increased caloric intake of anutritionally complete, palatable diet, as animals are fed adlibitum. Nutritional insufficiencies from a high-fat diet can place
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strain on the body's vital organs such as the liver, kidneys, or heart(28). Analysis of serum albumin, alkaline phosphatase (ALP),aspartate aminotransferase (AST), alanine transaminase (ALT),and iron levels in tumor-bearing mice fed the sHFLC diet for over
1month (Fig. 3D–H) showed normal levels in all enzymes tested.KD-fed animals had reduced serum albumin levels (Fig. 3D) andincreased serum ALP levels (Fig. 3E), which has been demon-strated in long-term KD-fed mice (30). Analysis of cholesterol,
Figure 1.Glucose restriction slows proliferation of patient-derived gliomaspheres. All gliomaspheres were cultured on a 7-day passage cycle. A, growth curve ofL0 patient-derived gliomaspheres cultured long-term [over a period of 6 passages (6 weeks)] in altered and normal glucose containing medias. Long-term growthcurve expressed in log-based on calculated fold expansion values for each serial passage. Fold expansion (F) is determined by dividing final density by platingdensity. B, average fold expansions of four patient-derived glioma spheres cultured in normal glucose media (NG: 500þ mg/dL), physiologic glucose media(PG: 90–110 mg/dL) and physiologic-low glucose media (LG: 60–85mg/dL) over a single 7-day passage cycle (n¼ 50 single passages). C, MTT enzymatic assay offour patient-derived gliomasphere cultures, expressed as a percent of control (NG - normal glucose media; n ¼ 3 assays, 6 wells per assay). Each line isstatistically comparedwith its control; no comparison between cell lines ismade. D, quantification of percent positive Ki-67 L0 cells grownover a 7-daypassage cyclemeasured by flow cytometry (n ¼ 10). E, quantification of percent positive MCM2 L0 cells grown over a 7-day passage cycle measured by flow cytometry(n¼ 10). F, quantification of percent positive caspase-3 L0 cells over a 7-day passage cyclemeasured by flow cytometry (n¼ 7). Overall one-wayANOVA significant(P ¼ 0.0483), Bonferroni post hoc comparison insignificant. G, single well image from a 96-well plate of L0 gliomaspheres cultured in altered and normal glucosecontaining medias. Gliomaspheres plated 250 cells per well and grown for 7 days, fixed and stained with 4% PFA/.01% DAPI and imaged using a Leica DMI 4000 Bmicroscope. White box in bottom right corner contains an enlarged gliomasphere. H, quantification of 96-well plates using Macnification to determine sphere-forming frequency expressed as a percentage of control [normal glucose containing media (NG)]; (n ¼ 20 wells). I, quantification of 96-well plates usingMacnification for average sphere diameter in um (n>500 spheres, 20wells). J, calculated KLL values. KLL¼ LN(F)/t, whereby F¼ fold expansion, division of cell countat end of passage/cell count at start of passage, and t ¼ time (days in culture). Statistics performed using a one-way ANOVA with Bonferroni post hoc test.� , P < 0.01; �� , P < 0.001; ��� , P < 0.0001 compared with control (NG). #, P < 0.01; ##, P < 0.001; ###, P < 0.001 compared with PG.
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triglycerides, HDL, LDL, and non-esterified fatty acids (NEFA)were also performed (Fig. 3I-M), as high cholesterol levels are aconcern with a high-fat diet (31). Reports of mice fed the Bioserv-KD describe increases in hepatic triglycerides and fat accumula-tion, together with a reduction in body weight, due to cholinedeficiency combined with low-protein and high-fat content (32).In line with these findings, animals fed the Bioserv-KD showed asignificant increase in cholesterol (Fig. 3I), triglycerides (Fig. 3J),HDL (Fig. 3K), and LDL levels (Fig. 3L). sHFLC-fed animalsshowed statistically significant high levels of HDL only(Fig. 3K). Together, these results indicate that the sHFLC diet isnutritionally safe.
The sHFLC diet slows tumor progression, increases survival,and reduces tumor burden in subcutaneous and orthotopicxenograft models
In our preclinical subcutaneous xenograft model, L0 GB cellswere grown until palpation (1�1 mm), at which time dietarytreatments were introduced. Overall tumor volume for the sHFLCand KD-fed mice were similar, showing a significant reduction intumor progression compared with controls (Fig. 4A and Supple-mentary Fig. S4A]. In addition, compared with control, bothtreatment groups demonstrated a significant delay in time toreach a tumor volumeof 50mm3; underwhich tumor progressionis not considered to have occurred (Fig. 4B). Both treatmentgroups also demonstrated a significant delay in time to reach1,766mm3 (15�15mm:endpoint; Fig. 4C). These results indicateincreased progression-free survival and time to reach endpoint inboth experimental groups. Similar results were obtained with L2,with respect to body weight, tumor progression, and delayedprogression to endpoint (Supplementary Fig. S3C and S4B andS4C]. Overall, the sHFLC diet demonstrated similar antitumorefficacy as compared with the KD, in a subcutaneous xenograftmodel of tumor progression.
We assessed the efficacy of the sHFLCdiet on tumor burden andhost survival using histologic techniques in our intracranialxenograft model. Brains from tumor-bearing animals werestained with H&E and human-specific Nestin and tumor burdenwas confirmed in all three groups (Fig. 4Dand Supplementary Fig.S4D–S4F]. Animals fed the sHFLC and KD demonstrated a 3-fold
reduction in tumor occupancy compared with controls (Fig. 4E).Brains from mice fed the control and sHFLC diets, when stainedwith active caspase-3 and Ki-67, showed no change in apoptosis,but did show a reduction in proliferation (Supplementary Fig.S4G).
It has been proposed that a potential combinatorial treatmentof metformin with carbohydrate restriction could result inenhanced antitumor efficacy (33). Mice fed the KD and sHFLCalone demonstrated a significant increase in survival comparedwith the control-fed mice (Fig. 4E and Supplementary Fig. S4H),statistically equal to metformin alone. In both xenograft models,metformin alone was able to reduce blood glucose, reduce tumorprogression, and increase survival, yet the combination sHFLCdiet andmetformin showed no additive or synergistic effects (Fig.4E; Supplementary Fig. S4I–S4K). These data show a lack ofcombinatorial efficacy, potentially stemming from an overlap-ping mechanism of action. Metformin is a well-characterizedactivator of AMP-activated protein kinase (AMPK), an enzymeresponsible for cellular energy homeostasis and negative regula-tion of themTORpathway. It is possible that the energy restrictionvia the sHFLC is also activating AMPK and, in turn, inactivatingthe mTOR pathway. These data indicate that the sHFLC diet iscapable of increasing animal survival while minimizing tumorburden, is as effective as metformin, and may mechanisticallyoverlap in AMPK-mediated inactivation of the mTOR pathway.
sHFLCnutritional intervention correlates with downregulationof the mTOR pathway in implanted GB cells
To determine the potential mechanistic effects of the sHFLCdiet, we analyzed 86 key genes in the mTOR pathway, throughrtPCR, using an mTOR Profiler Kit. Four control subcutaneoustumors and four sHFLC-fed subcutaneous tumors were analyzed.Overall, there was a global downregulation of mTORC1/mTORC2 genes in sHFLC-fed tumors compared with control(Fig. 5A), with an inverse relationship observed in negativepathway regulators. All significantly down-or-upregulated genesare shown in Fig. 5B and grouped as functional sets. Comparisonof regulatory genes in functional groups indicates that the mostaffected clusters are the positive mTORC1 regulators and thetranslational effectors downstream of mTORC1/2. Inhibition of
Figure 2.Mimicked sHFLC in vitro (low glucose and ketone treatment) further reduces the proliferation of patient-derived gliomaspheres. A, dose response curveof L0 patient-derived gliomaspheres treated with increasing concentrations of ketones (b-hydroxybutyrate, BHB), expressed via fold expansion. Assay performedin normal glucose-containing media, physiologic media, and low glucose media to determine EC50 value and optimal ketone treatment in vitro. The b-hydroxybutyrate (BHB) EC50 for NG¼ 15.937mmol/L, PG¼ 4.063mmol/L, and LG¼ 2.021 mmol/L. B, fold expansion of consecutive single-passage patient-derivedL0 and L2 cells treated with 4 mmol ketones (KTX) in combination with normal (NG) and altered glucose medias (physiological-PG and low-LG). Foldexpansion calculated by dividing final density by plating density. N ¼ 50þ passages. C, MTT enzymatic assay of patient-derived L0 and L2 treated with 4 mmolketones (KTX) in combination with normal (NG) and altered glucose medias (physiologic-PG and low-LG). Dual wavelength subtraction 750 � 595 OD.Expressed as a percentage of control gliomaspheres grown in normalmedia (NG).N¼ 3 assays/12wells per assay. D, quantification of percent positive Ki-67 L0 cellsgrown over a 7-day passage cycle in normal (NG) and altered glucose medias (physiologic-PG and low-LG), exposed to 4 mmol/L b-hydroxybutyratetreatment (KTX) measured by flow cytometry (n ¼ 10). E, quantification of percent positive MCM2 L0 cells grown over a 7-day passage cycle in normal (NG) andaltered glucose medias (physiologic-PG and low-LG), exposed to 4 mmol/L b-hydroxybutyrate treatment (KTX) measured by flow cytometry (n ¼ 10). F,quantification of percent positive active caspase-3 L0 cells grown over a 7-day passage cycle in normal (NG) and altered glucose medias (physiologic-PG andlow-LG), exposed to 4 mmol/L b-hydroxybutyrate treatment (KTX) measured by flow cytometry (n ¼ 10). G, DAPI-stained 96-well images of patient-derivedGBM cell line 0 grown in normal glucose containing media, physiologic media, and low glucose media with and without 4mmol/L ketone treatment. Gliomaspheresplated 250 cells per well and grown for 7 to 10 days, fixed and stained with 4% PFA/.01% DAPI and imaged using a Leica DMI 4000 B microscope. Whitebox surrounding enlarged gliomasphere in bottom right corner. H, quantification of 96-well DAPI-stained images using Macnification for sphere forming frequency,expressed as a percentage of the control (normal glucose grown cells) n ¼ 20 wells. I, quantification of 96-well DAPI stained images using Macnification foraverage sphere diameter (um). n ¼ 200þ cells. J, KLL Mathematical modeling of stem cell divisions, KLL ¼ LN(F)/t, whereby F ¼ fold expansion, division ofcell count at end of passage/ cell count at start of passage, and t ¼ time (days in culture). Statistics performed using a one-way ANOVA with Bonferronipost hoc comparative test. � , P < 0.01; �� , P < 0.001; ��� , P < 0.0001 when compared with NG control. #, P < 0.01; ##, P < 0.001; ###, P < 0.0001 when comparedwith physiologic control (PG). ^, P < 0.01; ^^, P < 0.001; ^^^, P 0.0001 when compared with low control (LG).
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both mTORC1/mTORC2 has been shown to be a potent anti-cancer therapy (34).
To investigate further the negatively affected mTOR effectorgenes, we assessed ribosomal S6, its activator p70S6K and mTORrelease of 4EBP1. In addition, AMPK activation and phosphory-lation of mTOR were assessed, as metformin primarily targetsAMPK. Flow analysis of multiple ex-vivo single-cell–dissociatedsHFLC-fed tumors confirmed a statistically significant reductionin the phosphorylation of ribosomal S-6 (Fig. 6A), the release ofthe translational repressor 4EBP1 (Fig. 6B) and a significantincrease in AMPK activation (Fig. 6C), when compared withcontrol. The proliferation marker MCM2 was also diminished in
all treatments groups (Fig. 6D). Control-fed tumors served aspositive mTOR controls with constitutive activity levels (PTENnegative; ref. 26), and metformin-fed tumors served as negativemTOR controls (AMPK activator; ref. 35. This was confirmedusing IHC analysis of subcutaneous tumors and xenograft mousebrains (Fig. 6E and F), as well as through Western blot analysis ofsubcutaneous tumors (Fig. 6G; targeting the S6 regulator p70S6K,p-mTOR, AMPK, and multiple epitopes of 4EBP1). The phos-phorylation of 4EBP1, and subsequent release of eIF4E, results ininitiation of translation, and modulation of this release directlyaffects tumorigenicity (36). Therefore, we conclude that thesHFLC diet can potentially diminish cellular proliferation
Figure 3.The sHFLC diet reduces blood glucose, increases blood ketones, maintains body weight, and is nutritionally complete. A, peripheral blood glucose levelsnormalized to bodyweight of animals on their respective diets, expressed as arbitrary units (mg/dL per gramof bodyweight). Blood glucose levels obtained using aPrecision Xtra One Touch glucose meter of animals fed control, sHFLC and ketogenic diets. Of note, 5 mL of blood drawn using tail snip method. Bloodglucose values can be found in Supplementary Fig. S3A. B, peripheral blood ketone body levels normalized to body weight of animals on their respective diets,expressed as arbitrary units (mmol per gram of body weight). Blood ketone body levels obtained using a Precision Xtra One Touch ketone meter of animalsfed control, sHFLC and ketogenic diets. Of note, 5 mL of blood drawn using tail snip method. Blood ketone values can be found in Supplementary Fig. SB. C,averaged animal body weight (g) over time. Weighted three times per week post-tumor palpation. D–M, serum blood analytes tested in tumor-bearing animals fedthe control, sHFLC and ketogenic diets. Blood obtained via retro orbital eye bleeds before sacrifice. Blood samples spun and serum separated. ComparativeClinical Pathology Services, LLC, carried out Blood analyte testing. � , P < 0.01; �� , P < 0.001; ��� , P < 0.0001 when comparing with control fed group. #, P < 0.01;##, P < 0.001; ###, P < 0.0001 when compared with the sHFLC; All statistics performed using a one-way or two-way ANOVA with Bonferroni post hoc test.
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through AMPK-mediated mTOR inactivation of 4EBP1 andthrough protein synthesis via ribosomal S6.
DiscussionWe report a dietary intervention that produces low circulating
glucose while elevating ketones and results in a substantialreduction inGB cellular proliferation.Maintaining in-vitro glucoseat levels representing physiologic (90–110mg/dL)or physiologic-lowbutnot hypoglycemic (65–80mg/dL), reduces the clonogenicfrequency of gliomaspheres, potentially affecting the cancerstem cell population. The addition of BHB also reduces GB
proliferation and when combined with reduced glucose levels,results in further reduction in proliferation. Here, we demonstratethat a high-fat, low-carbohydrate diet supplementedwithMCToil(sHFLC) is able to slow tumor progression and increase survival.In vivo, the sHFLC diet was similar to the ketogenic diet (KD) inantitumor efficacy, but showed nutritional advantages in bodyweight, organ enzyme levels, and lipid profile. Finally, we dem-onstrate that the sHFLC diet affects the mTOR signaling pathwayby reducing expression of upstream regulators and translationaldownstream effectors.
We utilized patient-derived GB stem cell lines that have beendemonstrated tonot only recapitulate the genotype andphenotype
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Figure 4.sHFLC diet slows tumor progression, increases progression-free survival, and overall survival while reducing tumor burden in a subcutaneous and orthotopicGB xenograft models. A, averaged overall tumor volume of animals with subcutaneous tumors fed either a control, sHFLC or ketogenic diet. Tumor calculationshown in Supplementary Fig. S4A. Tumors measured three times per week using calipers and calculated overall tumor volume averaged across all mice.B, delayed time progression to reach 50mm3 total tumor volume. C, delayed time progression to reach 1,766mm3 (endpoint) total tumor volume. D, H&E andNestin-stained whole brain OCT sections of mice fed a control, sHFLC diet or ketogenic diet (KD). Two control, two sHFLC-fed, and two KD-fed brains are illustrated in thetable. Human GBM cells were identified using anti-human Nestin antibody and visualized using the ABC-Elite peroxidase method (Vector Laboratories).Counterstaining of the nuclei was performed using hematoxylin. Additional anti-human Nestin fluorescent staining showed accompanying the permanent stainingto illustrate cellular invasion. E, analysis of cross-sectional area shows significantly decreased tumor occupancy for mouse brains fed the sHFLC diet andketogenic diet compared with controls. Graph shows average SE over 5 to 8 sections per group. P <0.0001. F, Kaplan–Meier survival curve ofmice transplantedwithL0 patient-derived GB treatedwith a control, sHFLC,metformin, sHFLCþmetformin, or ketogenic diet. � , P <0.01; �� , P <0.001; ��� , P <0.0001when comparingwithcontrol-fed group. Statistics performed for Kaplan–Meier using a Mandel-Cox, Nestin quantification using a Student t test and one-way ANOVA for tumorprogression.
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of the primary tumor (37), but also to be driven by a cancer stemcell population (38). They are grown in serum-free conditions,which serves as a clinically relevant model for studying GB in vitro
(39). Standard tissue culturemedia are supersaturatedwith glucoseto provide an excess of nutrients for continuous growth. However,available glucose is variable and highly dependent on nutritional
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Figure 5.The sHFLC diet diminishes mTORC1/2 signaling. A,clustergram of 78 selected mTOR-associated genesfrom Qiagen RT2Profiler with 86 key mTOR genes.Color gradient of clustergram shows consistentdownregulation of the mTOR pathway. Clustergramhas four different animal tumors from the control andsHFLC-fed groups. Tumors analyzed weresubcutaneously transplanted patient-derived line 0gliomaspheres. B, table of significant P values ofselected mTORC1/2 substrate genes grouped infunctional sets. Housekeeping geneswere determinedusing NormFinder and normalized using the Qiagenanalysis software. Statistics performed using aStudent t test.
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Figure 6.sHFLC potential mechanism of action. A–D, assessment of mTOR substrate activation via flow cytometry. Graphically represented as a percent of positivestaining based on control. Mean fluorescent intensity for each treatment group was averaged across multiple animal samples and normalized to be expressedas a percent compared with the normal fed control mice¼ 100%. E, immunofluorescent-stained subcutaneous xenograft tumor sections (5 mm) cryopreserved andembedded in OCT. Green ¼ antibody/Blue ¼ DAPI. All images were taken with a 10� lens on a Leica DM 4000 microscope. F, immunofluorescent-stainedmouse brain sections from intracranial xenograft (10�m) cryopreserved and embedded in OCT. Green¼ antibody/Blue¼ DAPI. All images were taken with a 10�lens on a Leica DM 4000 microscope. G, Western blot analysis for mTOR substrates using Metformin treatment as a positive control as well as mTORsiRNA. Quantification with ImageJ to the right. � , P < 0.01; �� , P < 0.001; ���, P < 0.0001 compared with control; #, P < 0.01; ##, P < 0.001; ###, P < 0.0001 comparedwith sHFLC; ^, P < 0.01; ^^, P < 0.001; ^^^, P < 0.0001 compared with KD. All statistics performed using one-way ANOVA with Bonferroni post hoc test.
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status or disease state in vivo. Excess glucose, as seen in GB patientswithpersistenthyperglycemia, leads topoorpatient survival (40). Ithas also been suggested that diets with a high glycemic index mayincrease the risk of tumorigenesis (41), and low-carbohydrate,high-protein diets that limit circulating glucose can delay cancerdevelopment and progression (42). In vitro, glucose withdrawaland treatment of GB cells with the non-metabolizing glucoseanalog 2-deoxy-glucose (2DG) results in up to 95% growth inhi-bition (43). These studies demonstrate the dependence of cancercells on glucose to promote survival and cellular growth. To assessthe reliance of our patient-derived GB lines on glucose, we reducedglucose concentrations. Using four GB lines, we show a significantdecrease in overall proliferation, an increase in apoptosis, andreductions in both clonogenic frequency and stem cell symmetricdivision rate when GBs are placed under glucose restriction (Fig. 1and Supplementary Fig. S1). These findings, together with otherpublished studies, support the hypotheses that high glucose con-tributes to tumor growth and that reducing glucose levels canattenuate tumor cell proliferation.
During periods of fasting or starvation, ketone bodies areproduced in the liver as compensatory energy sources, whereBHB/acetoacetates are metabolized into acetyl-coA and utilizedwithin the TCA cycle for oxidative ATP production (6). In contrastwith normal cells, GB cells have been shown to be incapable ofutilizing ketones as a metabolic substrate, due to a lack of 3-hydroxybutyrate metabolizing enzymes (44) and low expressionlevels of mitochondrial ketolytic enzymes (OXCT1 and BDH1;ref. 45). Neuroblastoma has also shown decreased expression ofsuccinyl-coenzyme A:3-oxoacid coenzyme A transferase (SCOT),a key enzyme in ketone body metabolism (46). In vitro treatmentof glioma cells with acetoacetate or BHB is able to delay both cellgrowth and metabolic enzyme activity (8), and reduces prolifer-ation and cell viability of VM-M3 cells (9). Other reports indicateBHB acts as an endogenous and specific inhibitor of class I histonedeacetylases (HDAC; ref. 47). HDAC inhibitors are an emergingantiproliferative, proapoptotic, and cell-cycle inhibiting antican-cer therapy (48). BHB treatment of HEK293 cells resulted in adose-dependent increase inhistone acetylation, even at 1-2mmol/L. In vivo, 24-hour–fasted or calorie-restricted animals showedincreased histone acetylation with ketone levels between 1.2 to1.5mmol/L (47). In addition, ketone body administration in vitrohas shown regulatory effects on glucose metabolism (49) andglycolysis (8). Therefore, BHB emerges as a novel environmental(dietary) regulator ofmetabolic health, chromatinmodifications,and epigenetic gene regulation (50). In contrast, Lisanti andcolleagues have reported in abreast cancermodel that high ketonelevels can promote tumor progression in-vivo and increasedmigration in vitro, but not in vivo (51). This study raises thepossibility that certain types of cancers, or biologic systems, maybe differentially affected by elevated ketones. Here, we show thattreatment of four GB stem cell lines, with increasing concentra-tions of BHB, caused a dose-dependent inhibition of prolifera-tion,with supra-physiologic concentrations (>40mmol/L), result-ing in almost no proliferation (Fig. 2A). Conversely, BHB treat-ment in escalating doses, as assessed by a MTT assay, indicated athreshold of anti-enzymatic activity, rather than a true doseresponse [Supplementary Fig. S2A—S2C]. We hypothesize thatthe previously discussed metabolic interaction of BHB may yielddifferential effects onGBproliferation and enzymatic activity. TheMTT assay measures NAD(P)H flux within the cell, which directlyrelates to metabolic activity, while cell numbers calculates fold
expansion. These different measurable outcomes indicate BHBtreatment has varying effects, potentially fromHDACor glycolysisinhibition, in cell populations that also differ in proliferation andmetabolic activity (Fig. 1B and C). Treatment of GB stem cell lineswith a constant physiologic concentration of BHB (4mmol/L), asseen in KDpatients, resulted in reduction of clonogenic frequencyand symmetrical stem cell divisions, suggesting that elevatedketones affect the putative cancer stem cell population (24). Thiseffect was seen in all patient-derived cell lines, which include theclassical (L0, L1, L2) and proneural (S3) subtypes of GB. GBsubtypes represent the variation in mutational status that mayaffect some aspect of metabolism and glycolysis. The proneuralsubtype exhibits isocitate dehydrogenase 1 (IDH1) mutations,which alter the cell's ability to regulate redox,NAD(P)H levels andmetabolize several macromolecules (52), potentially making thissubtype more susceptible to metabolic interventions.
The combination of reduced glucose and increased ketonebodies has shown an enhanced anticancer effect. The KD mimicsthese biologic effects and has been proposed as a treatment for GBand other cancers (5). This was first explored in two pediatricpatients with advanced-stage astrocytomas, following SOC treat-ments, and was able to produce long-term (>5 years) tumormaintenance, employing a KD supplemented with MCT oil(16). Since this landmark report, numerous groups have inves-tigated the antitumor efficacy of a KD and an RKD in several typesof cancer. RKD in experimentalmousemodels of glioma has beenshown to be antitumorigenic, antiangiogenic, and prosurvival(14), while also being anti-invasive, anti-inflammatory, andproapoptotic by targeting signaling pathways related to glucoseand glutamine metabolism (4). In animal models, feeding withtheKD ad libitumhas been reported to increase survival and reducetumor growth (11). Other preclinical animal models such asgastric cancer (53), colon cancer (54), and metastatic cancer(13) have used the KD, reporting similar antitumorigenic effects.Several case reports describe the applicability (20) and combina-torial safety of KD and RKDwith current SOC therapies (17), withsome patients experiencing stable disease on the diet and othersexperiencing unable tomaintain (19). In 2014, the first phase oneclinical trial assessing the safety of the KD on recurrent glioblas-toma determined that the diet is safe and feasible, warrantingother trials to explore the combinatorial potential of the KD withcytotoxic treatments (18). The KD in the context of cancer ispromising, however patient populations are diverse, as are theirdietary needs (21), creating a demand for alternative dietaryinterventions for tumor management (22).
We designed the sHFLC diet for long-term sustainable main-tenance of GB and for increased flexibility and palatability. Weutilized NOD/SCID xenografts in this study for the advantages instudying measureable outcomes of human tumor responses totherapy. It should be noted, however, that NOD/SCID micedisplay the effects of both type I and type II diabetes, are immu-nocompromised and have an altered metabolic state. Our pre-clinical models of tumor progression and survival support thefeasibility of the sHFLC diet, as we can effectively slow theprogression of GB and increase animal survival with diet alone(Fig. 4), using two patient-derived GB lines. Notably, sHFLC-fedanimals, compared with the Bioserv-KD, had superior nutritionalstatus demonstrated by increased body weight and improvedblood analyte and lipid profiles (Fig. 3). The Bioserv-KD weutilized contains large amounts of lard-based fat, and low levelsof protein and choline, which increases hepatic triglycerides and
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cholesterol while simultaneously reducing body weight (32).Furthermore, the 1:6 carbohydrate:fat KD utilized in this studyis higher in fat content than the KD typically administered topatients (1:4). Patients on a 1:4 or 1:3 KD can better manage fatsources to attenuate issues such as weight loss and increasedcholesterol, which have been demonstrated here with animalson a 1:6 KD and attenuated with animals on the sHFLC. Our GKIvalues were comparable to previous publications, but were moreindicative of an RKD than an ad libitum KD. However, whencomparing GKI values to human data, patients receiving a KDsupplemented with MCTs (also fed ad libitum) exhibit valuescloser to the ones we report. We hypothesize that the utilizationof MCTs, which inherently increase ketone body production invivo, may skew the GKI towards a more favorable value. MCTsupplementation has also been shown to inhibit proteolytic andlipolytic factors of cachexia and increase body weight in cachecticcancer patients (55), which is not typically a problem for braintumor patients but may still be useful for patients with othercancer types susceptible to metabolic intervention.
Pharmacologic intervention to temper circulating glucose hasbeen explored,with the assumption that a combinationofKDandmetformin could work together as a comprehensive anticancertherapy (33). We proposed to enhance our dietary paradigmwiththe addition of Metformin to the sHFLC diet (SupplementaryTable S1). Metformin alone significantly reduced tumor volumeand enhanced survival in two preclinical glioma models, indicat-ing metformin as a promising anticancer therapy. When metfor-min was combined with the sHFLC diet, there were no additive orsynergistic effects (Supplementary Fig. S4), indicating possibleoverlapping mechanisms of action. Metformin has multiple pro-posed antitumor mechanisms, but it is well characterized toactivate AMPK, a negative regulator of the mTOR pathway(33). The anticonvulsant mechanism of the KD is still unclear,however, one proposed mechanism is also inhibition of themTOR pathway (56, 57). Taking into account the proposed KDmechanism, the known metformin mechanism and the lack ofcombinatorial antitumor efficacy, we posit that a potential mech-anism of action for the sHFLC diet is through diminished mTORsignaling (Figs. 5 and 6). A large body of literature exists on themTOR pathway, its substrates and how its hypo/hyper-activationresults in various disease states (35). ThemTOR pathway is one ofthe largest and most utilized pathways in cellular signaling, withtwo complexes (mTORC1/mTORC2) that have demonstrated arole in tumorigenesis. Recently, it's been shown that inhibition ofboth mTORC1/mTORC2 signaling results in dramaticallyreduced cell viability in glioma cell lines, as well as inhibitionof tumor growth in vivo (34). In our assessment of the sHFLCdiet'seffects on the mTOR pathway, we found significant reduction inboth mTORC1/mTORC2 signaling (Fig. 5). In our preclinicalmodels, sHFLC-fed tumors showed significant reduction indownstream mTOR effector signaling, whereby reduction inphosphorylation of 4EBP1 and ribosomal S6 inhibits both trans-lation and protein synthesis needed for cellular replication (36)(Fig. 6). Taken together, these findings indicate that inhibition ofthe mTORC1/2 pathway can be achieved through dietary inter-vention, resulting in a potent anti-cancer treatment.
Our work demonstrates that there is a distinct relationshipbetween metabolism and proliferation that can be exploited bychanging the energy sources in the body. Further research intothe biochemical reactions of metabolic intermediates may shedmore light on how ketone bodies are differentially utilized bytumor cells, as the role of mitochondria in tumor propagationand carcinogenesis is multifaceted and incompletely under-stood. Nevertheless, we effectively show that a combinationof low glucose and high ketones results in negative proliferativeeffects on gliomaspheres, which can be translated in vivo withthe sHFLC diet. This diet reduces overall tumor burden andincreases survival, equivalent to a strict 1:6 KD, and has acomplete nutritional profile. Hence we propose that dietarytherapy, such as the sHFLC diet, could be utilized in themanagement of GB.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: R.T. Martuscello, V. Vedam-Mai, C.L. Skinner,L.P. Deleyrolle, B.A. ReynoldsDevelopment of methodology: R.T. Martuscello, C.L. Skinner, O. Suslov,L.P. Deleyrolle, B.A. ReynoldsAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.T. Martuscello, D.J. McCarthy, M.E. Schmoll,C.D. Louviere, B. Griffith, C.L. Skinner, B.A. ReynoldsAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R.T. Martuscello, V. Vedam-Mai, M.A. Jundi,C.D. Louviere, L.P. Deleyrolle, B.A. ReynoldsWriting, review, and/or revision of the manuscript: R.T. Martuscello,V. Vedam-Mai, L.P. Deleyrolle, B.A. ReynoldsAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R.T. Martuscello, D.J. McCarthy, C.D. Louviere,O. Suslov, L.P. Deleyrolle, B.A. ReynoldsStudy supervision: V. Vedam-Mai, L.P. DeleyrolleOther (Submitted by Regina Martuscello regarding all images in this man-uscript submission): B.A. Reynolds
AcknowledgmentsThe authors thank all members of the B.A.R lab and undergraduate
volunteers for their assistance, specifically, Andrew Naples, James McGui-ness, Lindsey Chason, Hunter Futch, and Gretter Diaz. The authors alsothank their funding sources and the McKnight Brain Institute core facilities offlow cytometry and the cell and tissue analysis core (CTAC), in addition tothe University of Florida College of Medicine and the InterdisciplinaryProgram in Biomedical Sciences, and Barbara Frentzen and the FCBTR forbrain tumor samples.
Grant SupportThis work was financially supported by McKnight Brain Institute, Depart-
ment of Neurosurgery (to B.A. Reynolds), Florida Center for Brain TumorResearch (to B.A. Reynolds), National Brain Tumor Society (to B.A. Reynolds),NIH – NINDS R24 NS086554-01 (to B.A. Reynolds), NIH/NCI - R21CA141020-01 (to B.A. Reynolds), and American Cancer Society Chris DiMarcoInstitutional Research Grant (to L.P. Deleyrolle).
Received April 15, 2015; revised October 30, 2015; accepted November 12,2015; published OnlineFirst December 2, 2015.
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www.aacrjournals.org Clin Cancer Res; 22(10) May 15, 2016 2495
Supp. High-fat Low-Carbohydrate Diet for Glioblastoma
2016;22:2482-2495. Published OnlineFirst December 2, 2015.Clin Cancer Res Regina T. Martuscello, Vinata Vedam-Mai, David J. McCarthy, et al. of GlioblastomaA Supplemented High-Fat Low-Carbohydrate Diet for the Treatment
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