Ketogenic diet enhances the anti-cancer effects of PD-L1 blockade in renal cell carcinoma

Abstract

Introduction: Clear cell renal cell carcinoma (ccRCC) is characterized by a predominant metabolic reprogramming triggering energy production by anaerobic glycolysis at the expense of oxydative phosphorylation. Ketogenic diet (KD), which consists of high fat and low carbohydrate intake, could bring required energy substrates to healthy cells while depriving tumor cells of glucose. Our objective was to evaluate the effect of KD on renal cancer cell tumor metabolism and growth proliferation.

Methods: Growth cell proliferation and mitochondrial metabolism of ACHN and Renca renal carcinoma cells were evaluated under ketone bodies (KB) exposure. In vivo studies were performed with mice (nude or Balb/c) receiving a xenograft of ACHN cells or Renca cells, respectively, and were then split into 2 feeding groups, fed either with standard diet or a 2:1 KD ad libitum. To test the effect of KD associated to immunotherapy, Balb/c mice were treated with anti-PDL1 mAb. Tumor growth was monitored.

Results: In vitro, KB exposure was associated with a significant reduction of ACHN and Renca cell proliferation and viability, while increasing mitochondrial metabolism. In mice, KD was associated with tumor growth reduction and PDL-1 gene expression up-regulation. In Balb/c mice adjuvant KD was associated to a better response to anti-PDL-1 mAb treatment.

Conclusion: KB reduced the renal tumor cell growth proliferation and improved mitochondrial respiration and biogenesis. KD also slowed down tumor growth of ACHN and Renca in vivo. We observed that PDL-1 was significantly overexpressed in tumor in mice under KD. Response to anti-PDL-1 mAb was improved in mice under KD. Further studies are needed to confirm the therapeutic benefit of adjuvant KD combined with immunotherapy in patients with kidney cancer.

Keywords: PDL1; adjuvant ketogenic diet; immunotherapy; metabolic reprogramming; mitochondrial biogenesis; renal cell carcinoma.

Figure 1

Ketone bodies reduced growth cell proliferation and increased mitochondrial respiration and biogenesis of ACHN cells. HG = 25mM glucose, 2mM L-glutamine, 10% non-dialyzed FBS, 0mM ßHB, 0mM AcAc and KB = 2.8mM glucose, 2mM L-glutamine, 10% non-dialyzed FBS, 5mM ßHB, 5mM AcAc (A) Quantification of ACHN cell growth exposed to KB compared to untreated cells (HG)for 6 days was assessed by Incucyte. (B) Glucose consumption and lactate production were assessed in cell supernatant from untreated HG-ACHN compared to KB-ACHN (n=4). (C) Ratio of lactate/glucose was estimated in supernatants from HG-ACHN and KB-ACHN (n=4). (D) Cytosolic NADH/NAD+ ratio was determined from the pyruvate/Lactate concentration ratio (n=4). Data are presented as mean ± SEM. Asterisks (*) indicate significant differences (p<0.05), (**) (p<0.001), (***) (p<0.0001). (E) ECAR production measurements (n=6).

Figure 2

Mitochondrial respiration and metabolism in ACHN cells exposed to KB. HG = 25mM glucose, 2mM L-glutamine, 10% non-dialyzed FBS, 0mM ßHB, 0mM AcAc and KB = 2.8mM glucose, 2mM L-glutamine, 10% non-dialyzed FBS, 5mM ßHB, 5mM AcAc (A) Oxygraphic measurements of mitochondrial basal respiration (n=10). (B) Cellular phosphorylating respiration measured in nmol O2/min/mg of protein (n=10). (C) Maximal cellular oxidative capacity determined with FCCP titration (n=10). (D) Quantification of mtDNA copy number in ACHN cells exposed to KB compared to untreated cells (n=4). (E) Citrate synthase (CS) measurement (n=6). (F) Gene expression of genes involved in mitochondrial biogenesis using qPCR in ACHN cells (n=4). (G) Quantification of fragmented, tubular and tube mitochondrial lengths (HG n=38; KB n=44). (H) Representative images of the mitochondrial network of ACHN exposed (right panel) or not to KB (left panel). Data are presented as mean ± SEM and asterisks (*) indicates significant differences (p ≤ 0.05), (**) (p ≤ 0.001), (***) (p ≤ 0.0001).

Figure 3

KD decreases tumor growth proliferation in vivo and increases gene expression of CD274. (A) Nude mice were grafted with ACHN cells at day 0 and treated with KD or not from day 7 to day 56. (B) Measurements of tumor volumes were assessed once a week during 8 weeks. (C) HES and immunohistochemical staining performed at day 56 to assess PD-L1 expression and Ki67 index in normal diet (upper panel) and ketogenic diet (downer panels) groups. Representative images are presented at objective x20 (a,b,g,h) x40 (c,d) and x10 (e,f). (a,b,c,d): HES staining showing undifferentiated high-grade tumor cells spindle-shaped with marked cytologic atypia. (e,f): Ki67 proliferative index (anti-mib1) was similar between the two groups regardless of the diet (g,h): PD-L1 expression was slightly increased in KD tumor cells. (D) Relative mRNA expression analyzed by RT-qPCR in tumor xenograft at day 56. (E) Relative mRNA expression analyzed by RT-qPCR in ACHN exposed or not to KB. Data are presented as mean ± SEM and asterisks (*) indicates significant differences (p ≤ 0.05).

Figure 4

Renca cells exposed to KB overexpressed CD274 while reducing cell number and increasing mitochondrial respiration. HG = 25mM glucose, 2mM L-glutamine, 10% non-dialyzed FBS, 0mM ßHB, 0mM AcAc and KB = 2.8mM glucose, 2mM L-glutamine, 10% non-dialyzed FBS, 5mM ßHB, 5mM AcAc (A, B) Renca cell number was estimated by crystal violet after being exposed to KB compared to untreated cells (HG for high glucose media) for 6 days (n=6). (C) Relative mRNA CD274 expression was analyzed by RT-qPCR Renca cells treated or not with KB (n=6). (D) Oxygraphic measurements of mitochondrial routine respiration (n=6). (E) Cellular phosphorylating respiration measured in nmol O2/min/mg of protein (n=6). (F) Maximal cellular oxidative capacity of Renca cells (n=6). (G) Glucose consumption and lactate production were assessed in cell supernatant from untreated HG-Renca compared to KB-Renca (n=4). (H) Ratio of lactate/glucose was estimated in supernatants from HG-Renca and KB-Renca (n=4) (I) ECAR production measurements (n=6). (*) indicates significant differences (p≤0.05), (**) (p≤0.001), (***) (p≤0.0001).

Figure 5

The efficacy of immunotherapy treatment is improved by adjuvant KD. (A) Balb/c mice were grafted with Renca cells at day 0 and treated with KD or not from day 12 to the end of the experiment. (B) Measurements of tumor volumes were assessed three times a week during 4 weeks for each group (10 mice per groups) exposed or not to KD and receiving anti-PDL1 mAb (αPDL1+ND or αPDL1+KD groups), isotype control (isotype+ND and isotype+KD groups) and PBS controls (vehicle+ND and vehicle+KD groups). (C) Comparison of tumor volume at day 18 between groups treated by anti-PDL1 and exposed to KD or ND. (D) Survival of mice exposed to KD or ND, treated with anti-PDL1 mAb or isotype control or PBS (vehicle) after grafting of Renca cells. (C) Statistical significance was determined using Mann-Whitney U test. Data are presented as mean ± SEM. (D) Survival curves were done using Kaplan-Meier method and compared using the Log-rank test. *P<0.05.