The efficacy of an unrestricted cycling ketogenic diet in preclinical models of IDH wild-type and IDH mutant glioma

Infiltrative gliomas are the most common neoplasms arising in the brain, and remain largely incurable despite decades of research. A subset of these gliomas contains mutations in isocitrate dehydrogenase 1 (IDH1mut) or, less commonly, IDH2 (together called “IDHmut”). These mutations alter cellular biochemistry, and IDHmut gliomas are generally less aggressive than IDH wild-type (IDHwt) gliomas. Some preclinical studies and clinical trials have suggested that various forms of a ketogenic diet (KD), characterized by low-carbohydrate and high-fat content, may be beneficial in slowing glioma progression. However, adherence to a strict KD is difficult, and not all studies have shown promising results. Furthermore, no study has yet addressed whether IDHmut gliomas might be more sensitive to KD. The aim of the current study was to compare the effects of a unrestricted, cycling KD (weekly alternating between KD and standard diet) in preclinical models of IDHwt versus IDHmut gliomas. In vitro, simulating KD by treatment with the ketone body β-hydroxybutyrate had no effect on the proliferation of patient-derived IDHwt or IDHmut glioma cells, either in low or normal glucose conditions. Likewise, an unrestricted, cycling KD had no effect on the in vivo growth of patient-derived IDHwt or IDHmut gliomas, even though the cycling KD did result in persistently elevated circulating ketones. Furthermore, this KD conferred no survival benefit in mice engrafted with Sleeping-Beauty transposase-engineered IDHmut or IDHwt glioma. These data suggest that neither IDHwt nor IDHmut gliomas are particularly responsive to an unrestricted, cycling form of KD.


Introduction
Diffusely infiltrative gliomas strike over 17,000 people in the United States per year [1]. The vast majority of these tumors recur and progress, despite advancements in surgery, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 chemotherapy, radiotherapy, and immunotherapy. In 20-39 year-olds, gliomas are the 2 nd most common cause of cancer death in men, and are the 5 th most common cause in women [1]. The most common type of primary brain cancer in adults is diffusely infiltrative glioma; the most common subtype of infiltrative glioma, glioblastoma (GBM), is unfortunately also the most lethal. Despite great advances in treating many other kinds of cancer, the median survival of GBM patients is still only 12-15 months after diagnosis, even with surgical resection, radiation, and temozolomide therapy [2,3]. Long-term prognosis is grim; only about 15% of patients with an infiltrative glioma survive 5 years after diagnosis. As a group, primary brain cancers rank #1 among all cancers in terms of average years of life lost [4]. Despite intensive research, very little progress has been made in the treatment of GBM, and new approaches are badly needed.
Alterations in cell metabolism have long been known to be a hallmark of cancer, ever since Otto Warburg first described the preferential reliance of cancer cells on aerobic glycolysis over oxidative phosphorylation [5,6]. However, it was only relatively recently that mutations in metabolic genes were found to occur in some cancers. For example, approximately 20-30% of infiltrative gliomas carry mutations in isocitrate dehydrogenase 1 (IDH1) or, far less commonly, IDH2 (together referred to as "IDH mut ") [7][8][9]. This subset of gliomas tends to occur in grade 2-3 gliomas, disproportionately arises in younger adults, and is associated with longer survival. Wild-type IDH1 and IDH2 encode enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate in the cytosol/peroxisomes and mitochondrion, respectively. In the process, these enzymes also generate reduced nicotinamide adenine dinucleotide phosphate (NADPH). Point mutations in codon 132 of IDH1 (usually R132H), and codon 172 of IDH2, cause the mutant enzymes to reduce α-ketoglutarate to D-2-hydroxyglutarate (D2HG), thereby consuming NADPH [10].
Because cancers mostly rely on glucose for energy and anabolism, numerous studies have explored the therapeutic potential of dietary carbohydrate restriction in cancer patients. This is achieved through a ketogenic diet (KD), which is characterized by high-fat, low-carbohydrate, and moderate-protein content. KD limits the bioavailability of carbohydrates and induces the liver to produce ketone bodies, such as β-hydroxybutyrate and acetoacetate, which are then converted into acetyl-CoA for use in the tri-carboxylic acid (TCA) cycle [11]. The goal of KD is to force tumor cells to use ketone bodies for energy, while still meeting the patient's basic nutritional needs.
KD has been tested as an adjuvant therapeutic strategy in a number of cancers, including GBM, with mixed results thus far [12,13]. However, an aspect of this research that has not yet been experimentally addressed is whether IDH mut gliomas might be particularly responsive to KD. One study suggested that the D2HG product of IDH mut can actually bind and inhibit ATP synthase, thereby inhibiting oxidative phosphorylation and ATP production [14]. In that study, human colorectal HCT116 cells transduced with IDH1 R132H were highly vulnerable to glucose deprivation in vitro, and were not able to use ketone bodies as effectively as IDH wt HCT116 cells. A recent clinical study found that 8 weeks of KD did indeed elevate urinary ketones in glioma patients, with no significant change in fasting glucose or hemoglobin A1c. Additionally, the levels of intratumoral ketones were similar between IDH wt and IDH mut glioma patients [15]. However, that study was not designed to evaluate antitumor efficacy. Therefore, we sought to explore whether KD might preferentially inhibit the growth of IDH mut gliomas in vitro and in vivo, using both patient-derived endogenous IDH1 wt and IDH1 mut xenografts in immunocompromised mice, as well as an isogenic Sleeping Beauty transposase-engineered model of IDH1 wt and IDH1 mut gliomas in immunocompetent mice.

Ethics statement
This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Northwestern University (protocol #5715), and allowed for flank tumors to reach a volume of 6,000 mm 3 (maximum dimensions of 20 mm x 30 mm). All surgery was performed under isoflurane inhalant anesthesia. Every effort was made to minimize animal suffering. Patient-derived cell lines were all developed under the auspices of an Institutional Review Board-approved protocol at Mayo Clinic and Duke University, with consent obtained from their donors.

Cell lines and cell culture
Five cell types were derived from the Mayo Clinic Brain Tumor Patient-Derived Xenograft National Resource [16]. Three were IDH1 wt glioblastomas (GBM6, GBM12, and GBM43), and 2 were IDH1 mut grade 4 astrocytomas (GBM164 and GBM 196). Two additional IDH1 mut cell types were TB09, a WHO grade 3 astrocytoma obtained from Dr Hai Yan at Duke University, and HT1080, a fibrosarcoma cell line from the American Type Culture Collection (the fibrosarcoma cell line was chosen to directly compare any possible differences in proliferation based solely on IDH1 mut status). All IDH1 mut cells were R132H except for HT1080, which was R132C IDH1. NPA and NPAC1 were rodent isogenic cell lines engineered using the Sleeping Beauty transposase system, gifted courtesy of Dr. Maria Castro from the University of Michigan [17]. Both NPA and NPAC1 have activating mutations in NRAS and inactivating mutations in TP53 and ATRX; NPAC1 also expresses IDH1 R132H [17]. All IDH1 mut cell types produced high amounts of D2HG via liquid chromatography-mass spectrometry, relative to the IDH1 wt cells (not shown). All cell types are authenticated via short tandem repeat analysis, and are summarized in Table 1.

In vitro proliferation
Cells were plated in 24 well plates at a concentration of 5x10 4 cells per well in triplicate. Experimental wells were supplemented with 10 mM β-hydroxybutyrate (Sigma Product #H6501). To mimic the low glucose environment characteristic of physiological ketosis, a formulation of DMEM (Corning) with 1.0 g/L of glucose was tested alongside the normal glucose concentration of 4.5 g/L. Plates were trypsinized at specific time points, and live cells were counted via trypan-blue exclusion using a BioRad TC20 Automated Cell Counter. Absolute cell counts were used to determine cell viability rather than the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay, as the latter uses mitochondrial metabolism as a marker of cell viability, and we sought to avoid any potential confounding effects of culture conditions on mitochondria.
In vivo studies GBM12, GBM164, and GBM196 cells were sourced from tumors propagated as subcutaneous growths in athymic nude mice and prepared for implantation. Briefly, after euthanizing each animal, tumors were aseptically excised from the flank and minced in a sterile culture dish with a scalpel. The cell suspension was centrifuged after mechanical disruption, filtered through 70 μM nylon-mesh filters, re-centrifuged, and re-suspended in an equal volume ratio of cell culture media to Matrigel. A 16-gauge needle and syringe were used to inject the cell suspension into the flanks of 6 week-old female NCr athymic nude mice (NCRNU-F sp/sp, Taconic). Intracranial injections of NPA and NPAC1 cells were performed as described previously in 10 week-old female C57BL/6J mice (Jackson) [17].
Animals received post-operative support care through administration of 0.9% saline solution, DietGel 76A, and thermal support while recovering from anesthesia until awake and ambulatory. One administration of meloxicam 1 mg/kg was given at the time of the procedure for 24 hour analgesia coverage, followed by another dose approximately 24 hours post-procedure, and a final dose 48 hours post-procedure if deemed necessary.

Animal monitoring and treatment
All animals were fed standard rodent chow (standard diet, or SD) for 3 days following flank or intracerebral engraftment before being randomized to either remain on SD ad libitum, or given a formulated KD (Ketogenic Diet TD.96355, Envigo Teklad Diets, Madison WI) ad libitum. The KD was received directly from the manufacturer and was a nutritionally complete diet composed of 15.3% protein, 0.5% carbohydrate, and 67.4% fat by weight (Table 2). Mice assigned to the KD group were kept on KD for one week, followed by SD for one week, and so on, in order to prevent obesity and reduce midlife mortality. Mice were still allowed to feed ad libitum, in order to reduce potentially confounding effects of weight loss from calorierestricted diets and more effectively maintain plasma ketone levels, as was described by others [13,18]. At the end of each week, blood from the saphenous vein was collected to test circulating metabolite levels using the Precision Xtra Blood Glucose and Ketone Monitoring System (Abbott SKU# 9881465). Animals were monitored twice weekly for signs of morbidity, such as weight loss, behavioral changes, and hunched positions, and were euthanized when tumor size

Statistical analyses
Differences between mean values of two groups were compared using unpaired two-sample ttests, or for interactions between glucose and ketones in vitro, via two-way ANOVA; P values less than 0.05 were considered significant. Log-rank tests compared survival between groups. Graph generation and statistical analyses were performed with GraphPad Prism 9 (GraphPad Software, San Diego, CA).

Results
First, we examined the effects of a ketogenic-like diet on cultured IDH1 wt and IDH1 mut patient-derived cancer cell lines under the following conditions: (i) normal basal glucose (NG) of 4.5 g/L; (ii) low glucose (LG) of 1.0 g/L; (iii) NG with 10 mM β-hydroxybutyrate (BHB); (iv) LG with BHB (Fig 1). Both pairs of IDH1 wt and IDH1 mut cell types responded similarly, with slightly attenuated proliferation in LG medium, but no effect of BHB in either NG or LG  Table 3.
https://doi.org/10.1371/journal.pone.0257725.g001 medium, as indicated by two-way ANOVA analyses (Table 3). Specifically, among IDH1 wt GBM6 cells, proliferation was higher in NG than LG ( Next, we evaluated the ability of an unrestricted, cycling KD (see "Methods") to induce ketosis in mice without tumors. Over the first 5 weeks that mice were on KD, blood glucose levels were similar to control mice on SD, even during weeks in which KD was implemented (Fig 2A). Interestingly, blood glucose rose 15% in KD mice after week 6 (147.8 mg/dl in KD versus 128.0 mg/dl in SD, P = 0.0029) (Fig 2A). This persisted in week 7 (144.3 mg/dl in KD versus 127.3 mg/dl in SD, P = 0.0053). Ketones, in contrast, increased by 112.5% after just one week on KD compared to mice on SD (1.13 mM versus 0.53 mM, P = 0.00022) (Fig 2B). While circulating ketones declined in KD mice during the weeks when they were back on SD, they remained 81-236% higher than in SD mice over the entire 7-week interval, consistent with previously published data by other who employed this type of KD [13,18]. Body mass was 13% higher in KD mice than SD mice by week 4 (26.3 grams versus 23.3 grams, P = 0.033), but this was transient, as the mass of the SD mice matched the KD mice by week 6 (Fig 2C).
Mice bearing subcutaneous flank patient-derived xenografts (PDX) of endogenous IDH1 wt GBM12, or IDH1 mut grade 4 astrocytoma (GBM164 and GBM196), were subjected to the unrestricted cycling KD or kept on regular SD. (The flank was chosen because IDH1 mut GBM164 and GBM196 cells grow poorly intracerebrally and in vitro, and these two PDX models were chosen for in vivo studies because IDH1 mut TB09 cells grow poorly in vivo.) As Table 3

Discussion
Altered metabolism in cancer cells raises the possibility of exploiting metabolic vulnerabilities to inhibit tumor growth, such as putting patients on KD. However, while KD may have efficacy against some cancers, clinical studies have been limited in glioma patients, and have so far mostly focused on feasibility, not outcomes [15,[21][22][23][24][25][26]. At the preclinical level, some studies showed that mice engrafted with IDH1 wt GBM did not benefit from KD [13,27], although others have suggested otherwise [28], and to the best of our knowledge, there has not yet been a direct experimental comparison of IDH1 wt and IDH1 mut gliomas exposed to KD. Another study by others suggested that IDH1 mut might confer sensitivity to KD, based on in vitro data using HCT116 cells engineered to express IDH1 R132H [14]. Thus, we sought to determine KD efficacy in a variety of preclinical in vitro and in vivo models of IDH1 wt and IDH1 mut glioma. Our data suggest that an unrestricted cycling KD does not have significant activity against either IDH wt or IDH mut glioma.
Since the original studies describing IDH1 mut and its neoenzymatic activity in cancer, a great deal of research has been done studying the metabolic effects of IDH1 mut , often generating conflicting results. For example, while some have shown that IDH1 mut depletes the cell of TCA intermediates [29][30][31][32][33][34], others have found little to no changes in those intermediates [10,35,36]; indeed, IDH1 mut gliomas may actually use lactate and glutamate anaplerosis to replenish TCA intermediates [35]. Some have suggested that glycolysis is reduced in IDH mut gliomas [34,36], but one group found reduced glucose uptake by IDH mut glioma cells, and otherwise no difference in the rate of glycolysis compared to IDH wt cells [37].
Results in IDH1 mut metabolic research seem to vary greatly depending on whether one is studying cells artificially overexpressing IDH mut , or is focusing on cells and tissues with endogenous IDH mut , as more pronounced metabolic changes tend to occur in the former than the latter [37]. This suggests that cells with naturally-occurring IDH mut may, over time, adjust their metabolism to at least partially compensate for perturbations caused by the mutant enzyme. For example, IDH1 mut gliomas may adjust for the depletion of TCA intermediates by upregulating glutamate dehydrogenase 2 expression [34]. Since IDH mut gliomas mostly use the TCA precursor glutamine to produce D2HG, these tumors compensate by turning pyruvate into TCA chemicals [38,39]. Although IDH mut consumes NAPDH, which should lead to glutathione depletion, IDH mut gliomas upregulate enzymes involved in glutathione synthesis, thereby maintaining glutathione levels [40]. Thus, when IDH wt wild-type cells are abruptly forced to overexpress IDH mut , any metabolic results, including sensitivity to KD-like conditions, need to be validated in patient-derived and/or transgenic models with endogenous IDH mut . Just as there is heterogeneity in metabolism-themed IDH mut research, a variety of KD regimens have been studied in preclinical models of disease. Two major sources of such variation include ad libitum versus caloric restriction, and cycling versus non-cycling KD. In one study combining non-cycling KD and caloric restriction in mice intracranially engrafted with syngeneic IDH wt CT-2A gliomas, caloric restriction on a regular diet reduced tumor growth, but the addition of KD to the caloric restriction had no significant effect [41]. Another study from the same group showed that a different calorie-restricted, non-cycling KD slowed the in vivo growth of IDH wt CT-2A mouse glioma cells and IDH wt U87-MG human glioma cells [42]. However, a different group reported that an unrestricted, non-cycling KD was able to slow the in vivo growth of isogenic GL-261 mouse glioma cells [43,44], and such diets do lead to an accumulation of BHB within engrafted tumors [28,43]. In a nontumor preclinical model of aging, an unrestricted cycling KD reduced midlife mortality and improved memory in C57/ BL6 mice [18]. Since that type of KD had never been tried in animal models of gliomas, and prolonged adherence to KD is notoriously difficult [15,45], we tested that same unrestricted cycling KD regimen in both patient-derived and engineered mouse glioma cells, with negative results in both settings.
A recent patient-based study showed that KD causes similarly elevated levels of ketones within IDH wt and IDH mut gliomas by magnetic resonance spectroscopy [15], but patient survival was not a part of that analysis. Other recent preclinical and clinical studies have suggested that gliomas can metabolically adapt to ketogenesis, and even use ketones to facilitate disease progression [25,27]. Our in vitro and in vivo experimental results align with such studies, and show that, despite achieving a ketosis-like state in tumor bearing animals, no difference in overall tumor volume or survival benefit was observed. Thus, our data suggest that an unrestricted cycling KD does not have a role in glioma patients, regardless of IDH1 mut status.