Ketogenic diet and chemotherapy combine to disrupt pancreatic cancer metabolism and growth

Abstract

Background: Ketogenic diet is a potential means of augmenting cancer therapy. Here, we explore ketone body metabolism and its interplay with chemotherapy in pancreatic cancer.

Methods: Metabolism and therapeutic responses of murine pancreatic cancer were studied using KPC primary tumors and tumor chunk allografts. Mice on standard high-carbohydrate diet or ketogenic diet were treated with cytotoxic chemotherapy (nab-paclitaxel, gemcitabine, cisplatin). Metabolic activity was monitored with metabolomics and isotope tracing, including 2H- and 13C-tracers, liquid chromatography-mass spectrometry, and imaging mass spectrometry.

Findings: Ketone bodies are unidirectionally oxidized to make NADH. This stands in contrast to the carbohydrate-derived carboxylic acids lactate and pyruvate, which rapidly interconvert, buffering NADH/NAD. In murine pancreatic tumors, ketogenic diet decreases glucose's concentration and tricarboxylic acid cycle contribution, enhances 3-hydroxybutyrate's concentration and tricarboxylic acid contribution, and modestly elevates NADH, but does not impact tumor growth. In contrast, the combination of ketogenic diet and cytotoxic chemotherapy substantially raises tumor NADH and synergistically suppresses tumor growth, tripling the survival benefits of chemotherapy alone. Chemotherapy and ketogenic diet also synergize in immune-deficient mice, although long-term growth suppression was only observed in mice with an intact immune system.

Conclusions: Ketogenic diet sensitizes murine pancreatic cancer tumors to cytotoxic chemotherapy. Based on these data, we have initiated a randomized clinical trial of chemotherapy with standard versus ketogenic diet for patients with metastatic pancreatic cancer (NCT04631445).

Conflict of interest statement

DECLARATION OF INTERESTS J.D.R. is an adviser and stockholder in Kadmon Pharmaceuticals, Colorado Research Partners, L.E.A.F. Pharmaceuticals, Bantam Pharmaceuticals, Barer Institute, and Rafael Pharmaceuticals; a paid consultant of Pfizer; a founder, director, and stockholder of Farber Partners, Serien Therapeutics, and Sofro Pharmaceuticals; a founder and stockholder in Toran Therapeutics; inventor of patents and patent applications held by Princeton University, including a patent application related to ketogenic diet for cancer therapy.

Figures

Figure 1.. Circulating 3-hydroxybutyrate, unlike circulating lactate, unidirectionally delivers hydride to tissues

(A) Illustration of exchange between circulatory lactate with tissue pyruvate and circulatory 3-hydroxybutyrate with tissue acetoacetate. 2H-tracers report on the exchange flux shown in the lactate schematic (and not in the 3HB schematic, because such exchange does not occur). Experimentally, whole-body exchange flux is the difference between 2H-tracer turnover flux and 13C-tracer turnover flux. Red, 2H (deuterium); blue, 13C. (B) Circulatory turnover flux (units of nmol/min/g body weight) for 2H- versus 13C-lactate in healthy mice fed standard diet. Greater 2H than 13C flux indicates reversible hydride exchange. Mean ± SEM, n = 5 for 2H-lactate, n = 10 for 13C-lactate. *p < 0.05 by two-tailed Student’s t test. (C) Circulatory turnover flux for 2H- versus 13C-3HB in healthy mice fed standard diet. Equal turnover flux indicates unidirectional catabolism into acetoacetate. Mean ± SEM, n = 5 for 2H-3HB, n = 7 for 13C-3HB. (D) Mass isotope distribution of KPC tumor chunk allograft lactate and pyruvate following [U-13C]lactate infusion for mice fed standard diet. Identical labeling patterns are consistent with rapid interconversion of these metabolites. Mean ± SEM, n = 6. (E) Mass isotope distribution of tumor 3HB and acetoacetate following [U-13C]3HB infusion for mice fed standard diet. Lack of acetoacetate labeling reflects its production from sources other than circulating 3HB. Mean ± SEM, n = 6. See also Figure S1

Figure 2.. Ketogenic diet suppresses glucose metabolism and induces ketone body oxidation in KPC tumors (A–G, tumor chunk allograft; H, primary)

(A) Metabolite concentrations of serum and bulk KPC tumors in mice fed with control or ketogenic diet. Mean ± SEM, n ≥ 5. (B) Serum insulin and c-peptide level in mice fed with control or ketogenic diet. Mean ± SEM, n = 4 for insulin, n = 6 for c-peptide. (C) Intratumor redox pairs in mice fed with control or ketogenic diet. Mean ± SEM, n = 17. (D) Tumor versus circulating lactate labeling from [U-13C]glucose and [U-13C]lactate infusion. In ketogenic diet, tumor lactate comes more from circulating lactate, as opposed to from intratumoral glycolysis. Mean ± SEM, n = 3. (E) Tumor metabolite labeling from [U-13C]3HB infusion. Tumor metabolite enrichment was normalized to the enrichment of circulatory [U-13C]3HB. Mean ± SEM, n = 3 for control diet, n = 4 for ketogenic diet. (F) Direct contributions of circulating nutrients to tumor acetoacetate. Mean ± SD. (G) Direct contributions of circulating nutrients to tumor TCA cycle (malate as representative metabolite). Mean ± SD. (H) MALDI-imaging mass spectrometry of malate labeling from 13C-glucose (top) and 13C-3HB (bottom) in pancreas from KPC mice. Fractional enrichment indicates 13C enrichment normalized to the 13C enrichment of circulatory infusates. T, tumor; F, peritumoral fibrosis. *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed Student’s t test. n.s., not significant. See also Figure S2.

Figure 3.. Ketogenic diet synergizes with cytotoxic chemotherapy

(A) Chemotherapy regimen (triple chemotherapy: nab-paclitaxel, gemcitabine, cisplatin) with doses administered on day 4 and day 7 after diet switch in C57BL/6 mice with KPC tumor chunk allografts. (B): Tumor volumes for diet only without chemotherapy (batch #2, mean ± SEM, n = 8 mice per group). (C) Kaplan-Meier curve for diet only without chemotherapy (n = 14 mice per group). (D) Allograft tumor volumes in mice treated with control diet plus chemotherapy or ketogenic diet plus chemotherapy (batch #2, mean ± SEM, n = 11 mice per group; for other batches, see Figure S3B). (E) Kaplan-Meier curve for mice treated with control diet plus chemotherapy or ketogenic diet plus chemotherapy (n = 31 mice for Ctrl + Chemo and n = 35 mice for Keto + Chemo). (F) Representative H&E staining, cleaved caspase-3 staining, and Ki67 staining from tumor tissue treated with control diet plus chemotherapy or ketogenic diet plus chemotherapy. Scale bar, 50 μm. (G) Quantification of cleaved caspase-3-positive cells and Ki67-positive cells within tumor tissues from (F) (n = 10 images for Ctrl + Chemo and n = 10 images for Keto + Chemo group). (H) Primary tumor volumes in KPC genetically engineered mouse model (GEMM) (each line represents one mouse). (I) Kaplan-Meier curve for KPC GEMM (n = 5 for Ctrl + Chemo group, n = 4 for Keto + Chemo group). *p < 0.05, ***p < 0.001. Two-way ANOVA in (B); log-rank (Mantel-Cox) test in (C), (E), and (I); two-tailed Student’s t test in (G). n.s., not significant. See also Figure S3.

Figure 4.. Ketogenic diet and classical chemotherapy disrupt redox homeostasis

(A) NADH/NAD ratio in KPC allografttumors (mean ± SEM, n = 17 for Ctrl group, n = 12 for Keto group, n = 17 for Ctrl + Chemo group, n = 14 for Keto + Chemo group). (B) Correlation of intratumor NADH/NAD ratio with tumor growth suppression. Mice were sacrificed on day 8, and NADH/NAD measured and plotted versus tumor growth (or, for values *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA with Tukey’s test in (A), (C) to (F); Pearson correlation in (B); two-tailed Student’s t test in (G).

Figure 5.. Ketogenic diet induces inflammatory gene expression in the tumor

(A) Gene expression in allograft KPC tumors of mice fed control or ketogenic diet. Ketogenic diet increases expression of genes related to the immune response and reduces expression of genes related to cholesterol synthesis. (B) Gene set enrichment analysis highlights increased pro-inflammatory genes in tumors of mice fed ketogenic diet. FDR, false discovery rate. See also Figure S5.

Figure 6.. Ketogenic diet-chemotherapy synergy does not require the adaptive immune system

(A) Chemotherapy regimen (nab-paclitaxel, gemcitabine) for immunocompromised NSG mice with KPC tumor allografts. (B) Tumor volumes in NSG mice (mean ± SEM, n = 12 for control diet + nab-paclitaxel + gemcitabine, n = 11 for ketogenic diet + nab-paclitaxel + gemcitabine). (C) Kaplan-Meier curve for NSG mice (n = 17 mice per group). (D) Tumor glycolytic metabolite levels. (E) Tumor nucleotide levels. (F) Tumor NADPH, NADP, and their ratios. (G) Tumor GSH, GSSG, and their ratios. In (D) to (G), n = 16 for control diet + nab-paclitaxel + gemcitabine, n = 11 for ketogenic diet + nab-paclitaxel + gemcitabine. *p < 0.05, **p < 0.01, ***p < 0.001. Two-way ANOVA in (B); log-rank (Mantel-Cox) test in (C); two-tailed Student’s t test in (D) to (G). See also Figure S6.