Fasting-Mimicking Diet Is Safe and Reshapes Metabolism and Antitumor Immunity in Patients with Cancer

Abstract

In tumor-bearing mice, cyclic fasting or fasting-mimicking diets (FMD) enhance the activity of antineoplastic treatments by modulating systemic metabolism and boosting antitumor immunity. Here we conducted a clinical trial to investigate the safety and biological effects of cyclic, five-day FMD in combination with standard antitumor therapies. In 101 patients, the FMD was safe, feasible, and resulted in a consistent decrease of blood glucose and growth factor concentration, thus recapitulating metabolic changes that mediate fasting/FMD anticancer effects in preclinical experiments. Integrated transcriptomic and deep-phenotyping analyses revealed that FMD profoundly reshapes anticancer immunity by inducing the contraction of peripheral blood immunosuppressive myeloid and regulatory T-cell compartments, paralleled by enhanced intratumor Th1/cytotoxic responses and an enrichment of IFNγ and other immune signatures associated with better clinical outcomes in patients with cancer. Our findings lay the foundations for phase II/III clinical trials aimed at investigating FMD antitumor efficacy in combination with standard antineoplastic treatments. SIGNIFICANCE: Cyclic FMD is well tolerated and causes remarkable systemic metabolic changes in patients with different tumor types and treated with concomitant antitumor therapies. In addition, the FMD reshapes systemic and intratumor immunity, finally activating several antitumor immune programs. Phase II/III clinical trials are needed to investigate FMD antitumor activity/efficacy.This article is highlighted in the In This Issue feature, p. 1.

Figure 1.

The fasting-mimicking diet (FMD) reduces blood glucose and growth factor levels in patients with cancer. A, Schematics of the FMD regimen, with the calorie content of each day of FMD (d1–d5). Black arrows indicate time points of blood/urine sample collection. Blood samples collected before the initiation of the FMD are indicated as “Pre,” whereas samples collected after the end of five-day FMD (i.e., before the initiation of refeeding) are indicated as “Post.” B, Bar plots indicating the number of patients (y-axis) who completed the number of FMD cycles indicated on the x-axis. C, Concentration of plasma glucose (mg/dL), serum insulin (10*μUI/mL), serum IGF1 (ng/mL), and urinary ketone bodies (mg/dL) before (Pre) and after (Post) FMD (first cycle) in 99 patients with cancer. D, Plasma glucose concentration (mg/dL) before and after FMD (across eight cycles; C1–C8). E, Serum insulin concentration (μUI/mL) before and after FMD (across eight cycles; C1–C8). F, Serum IGF1 concentration (ng/mL) before and after FMD (across eight cycles; C1–C8). G, BMI before and after FMD cycles (C1–C8) in the whole patient population. H, BMI before and after FMD (C1–C8) in patients undergoing FMD and cytotoxic chemotherapy (ChT). I, BMI before and after FMD (C1–C8) in patients undergoing FMD in combination with treatment other than ChT. All P values were determined by paired Wilcoxon test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. All comparisons for which the P value is not indicated did not show statistically significant differences (P ≥ 0.05). Data are represented as box plots showing median values, with the boundaries of the rectangle representing the first and third quartiles, while whiskers extend to the extreme data points that are no more than 1.5 times the interquartile range.

Figure 2.

The fasting-mimicking diet (FMD) reduces peripheral blood immunosuppressive cells and increases effector cells in patients with cancer and in healthy volunteers. A, Frequencies of CD14⁺, CD14⁺HLA-DR⁻, CD14⁺PD-L1⁺, and CD15⁺ cells before (Pre) and after (Post) FMD (first cycle) in 38 patients with different tumor types enrolled in the NCT03340935 trial. CD14⁺ and CD15⁺ cells were calculated as frequencies in total PBMCs (after debris and doublet exclusion), while CD14⁺PD-L1⁺ and CD14⁺HLA-DR⁻ cells were calculated as frequencies in total CD14⁺ cells. B, Frequencies of CD14⁺, CD14⁺HLA-DR⁻, CD14⁺PD-L1⁺, and CD15⁺ cells before (Pre) and after (Post) FMD (first cycle) in 13 patients with advanced breast cancer enrolled in the NCT03340935 trial and treated with first-line ChT plus FMD. C, Frequencies of CD14⁺, CD14⁺HLA-DR⁻, CD14⁺PD-L1⁺ and CD15⁺ cells before (Pre) and after (Post) ChT in a cohort of 13 patients with advanced breast cancer treated with ChT without the FMD. D, Frequencies of CD14⁺, CD14⁺HLA-DR⁻, CD14⁺PD-L1⁺, and CD15⁺ cells before (Pre) and after (Post) FMD in 8 healthy volunteers. E, Frequencies of pre- and post-FMD CD8⁺PD1⁺CD69⁺ (among CD3⁺CD69⁺), CD3⁺CD25⁺ (among CD3⁺), and CD3⁻CD16⁺CD56^dim (among CD3⁻) cells in 38 patients with different tumor types enrolled in the NCT03340935 trial. F, Frequencies of pre- and post-FMD CD8⁺PD1⁺CD69⁺ (among CD3⁺CD69⁺), CD3⁺CD25⁺ (among CD3⁺) and CD3⁻CD16⁺CD56^dim (among CD3⁻) cells in 13 patients with advanced breast cancer enrolled in the NCT03340935 trial and treated with first-line ChT plus FMD. G, Frequencies of CD8⁺PD-1⁺CD69⁺ (among CD3⁺CD69⁺), CD3⁺CD25⁺ (among CD3⁺), and CD3⁻CD16⁺CD56^dim (among CD3⁻) cells before and after ChT in 13 patients with advanced breast cancer treated with ChT without the FMD. H, Frequencies of pre- and post-FMD CD8⁺PD-1⁺CD69⁺ (among CD3⁺CD69⁺), CD3⁺CD25⁺ (among CD3⁺), and CD3⁻CD16⁺CD56^dim (among CD3⁻) cells in 8 healthy volunteers. I, Plasma concentration of CCL2, G-CSF, IL6, and IL8, as evaluated at the initiation and at the end of the FMD, in 34 patients with different tumor types enrolled in the NCT03340935 trial. CCL2, C-C motif chemokine ligand 2. All P values were determined by paired Wilcoxon test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. All comparisons for which the P value is not indicated did not show statistically significant differences (P ≥ 0.05). Box plots indicate median values of the indicated variable, with the boundaries of the rectangle representing the first and third quartiles, while vertical black lines extend to the extreme data points that are no more than 1.5 times the interquartile range. Each red dot represents one patient; couples of dots connected by the same black line refer to data from the same patient before and after the FMD.

Figure 3.

The fasting-mimicking diet (FMD) reshapes intratumor immunity in patients with breast cancer. A, Time points of blood and tissue sample collection in the DigesT trial (NCT03454282). T1, initiation of the FMD; T2, end of the FMD; T3, surgery; T4, approximately 30 days after surgery. B, Left, IHC evaluation of intratumor IGFR1 and phosphorylated IGFR1 (pIGF1R; brown dots) in pre- and post-FMD tumor samples from one indicative patient (magnification of 200×). B, Right, Box plots showing results of IHC analyses (reported as H-score) of IGF1R and pIGF1R in matched pre- and post-FMD tumor samples in 18 patients enrolled in the DigesT trial (NCT03454282). All P values for the indicated comparisons were determined by paired Wilcoxon test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Box plots indicate median values, with the boundaries of the rectangle representing the first and third quartiles, while vertical black lines extend to the extreme data points that are no more than 1.5 times the interquartile range. Each red dot represents one patient; couples of plots connected by the same black line refer to the same patient before and after the FMD. C, Representative images of hematoxylin and eosin staining (H&E) and IHC evaluations of intratumor CD8⁺ T cells and CD68⁺ macrophages in pre-FMD (T1) and post-FMD (T3) tumor samples from two representative patients (magnification of ×200). D, Top plots, box plots showing the IHC quantification of percentage (area) and absolute average numbers (per high-power field; HPF) of intratumor stromal CD8⁺ cells, absolute average number (per HPF) of intraepithelial CD8⁺ cells, absolute average number (per HPF) of CD68⁺ macrophages, and the ratio between stromal CD8⁺ cells (absolute average number per HPF) and CD68⁺ macrophages (absolute average number per HPF) in pre-FMD versus post-FMD tumor specimens. Box plot showing the CD8A/CD68 transcript ratio in post-FMD as compared with pre-FMD tumor specimens. Box plots indicate median values of the frequencies of each immune cell population/gene transcript, with the boundaries of the rectangle representing the first and third quartiles, while vertical black lines extend to the extreme data points that are no more than 1.5 times the interquartile range. Each red dot represents one patient; couples of dots connected by the same black line refer to data from the same patient pre- and post-FMD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by paired Wilcoxon test for the indicated comparisons. D, Bottom plots, correlation between CD8⁺ T cells by IHC and CD8A gene expression, and between CD68⁺ macrophages by IHC and CD68 gene expression; box plot showing the CD8A/CD68 transcript ratio in post-FMD as compared with pre-FMD tumor specimens. E, Heat map displaying scores of enrichment of leukocyte subsets (XCell, Charoentong list) that are differentially modulated after the FMD [T3 vs. T1, paired Wilcoxon test, P < 0.05, Benjamini–Hochberg (B-H) false discovery rate (FDR) < 0.1]. aDC, activated DCs; CD4⁺ and CD8⁺ Tcm: CD4⁺ and CD8⁺ central memory T lymphocytes; NKT, natural killer T cells; pDC, plasmacytoid dendritic cells; Tem, effector memory T lymphocytes. F, Box plots representing scores of individual leukocyte subsets modulated in post-FMD versus pre-FMD tumor specimens. G, Matrix correlation displaying Spearman correlation coefficients between RNA-seq data (CD8A and CD68 transcripts, CD8⁺ T-cell, and Macrophage enrichment scores), and IHC assessment of CD8⁺ T cells and CD68⁺ cells.

Figure 4.

Intratumor transcriptomic changes induced by fasting-mimicking diet (FMD). A, Volcano plots displaying differentially expressed genes comparing post-FMD (T3) versus pre-FMD (T1) gene-level RNA-seq data using negative binomial distribution (Deseq2 package). Benjamini–Hochberg (B-H) false discovery rate (FDR) and Log₂ fold change are represented. Left, red and blue colors are used to display upregulated and downregulated transcripts in post-FMD versus pre-FMD tumors, respectively; immune-related genes (see Methods and Supplementary Table S10) are colored in dark red (upregulated in post-FMD versus pre-FMD comparison) and dark blue (downregulated in post-FMD versus pre-FMD comparison); all the immune-related genes with −log₁₀ FDR > 2.5 are labeled; additional representative immune-related genes with −log₁₀ FDR > 1 are labeled; in the middle and left panels, transcripts belonging to the IFN-activating signature (IFNG.GS) and IFN-resistant signature (ISG.RS) are colored in purple and green, respectively. Volcano plot y-axis is truncated at −log₁₀ FDR = 10, as no immune-related genes above that cutoff were present. B, Heat maps representing differentially expressed genes in post-FMD (T3, surgical samples) versus pre-FMD (T1, tumor biopsies) comparisons; the top panel displays genes with P < 0.05 (Wilcoxon test) and B-H FDR < 0.1 at differential gene expression analysis and included in any of the significantly modulated (P < 0.05 and B-H FDR < 0.1) prognostic/predictive signatures at single-sample gene set enrichment analysis (of the 25 evaluated prognostic/predictive signatures, 13 were significantly enriched in post-FMD vs. pre-FMD tumor samples, but one of them did not yield any differentially expressed genes, therefore only 12 signatures are represented in the heat map; Supplementary Table S10); the middle panel displays genes with P < 0.05 (Wilcoxon test) and B-H FDR < 0.1 at differential gene expression analysis and included in the exhaustion and immune checkpoint list (Supplementary Table S10); the bottom panel displays genes with P < 0.05 and B-H FDR < 0.1 at differential gene expression analysis and included in the cytokine/chemokine gene list (Supplementary Table S10). In the heat maps, all genes with B-H FDR < 0.05 are labeled, in addition with other selected genes (in brackets) with FDR between 0.1 and 0.05. C, Box plots of representative prognostic/predictive signature enrichment scores and transcripts with P < 0.05 and B-H FDR < 0.1 in the post-FMD (T3) versus pre-FMD (T1) comparison. D, Box plots of Perforin 1 and Granzyme B IHC scores in post-FMD (T3) versus pre-FMD (T1) tumors (n = 17 and n = 16, respectively).

Figure 5.

Dynamic effects of FMD on systemic immunity and correlation with transcriptional immune modulation at tumor site. High-dimensional flow cytometry was performed in PBMCs from 19 of 22 patients enrolled in the DigesT trial selected for tumor RNA-seq analysis. A, Samples for high-dimensional flow cytometry were collected before the FMD (T1) and at different time points after the FMD (T2, T3, T4). We assessed the expression of multiple lymphoid and myeloid markers to define a total of 120 cell subsets (see Supplementary Fig. S11–S13). The nonparametric paired Wilcoxon test was used to compare the diverse immune cell frequencies at T2 versus T1, T3 versus T1, and T4 versus T1; 57 PBMC subpopulations undergoing modifications passing the significance cutoff at least at one time point (P < 0.05, Benjamini-Hochberg FDR < 0.1) are represented in the heat map. B, Loess regression curves of representative immune cell subsets are shown to illustrate modulation across time. See Supplementary Fig. S14 for the representation of the remaining PBMC Loess regression curves related to the significantly modulated PBMC subsets. C, Heat map representing Spearman correlations between significantly modulated blood PBMC subpopulations (n = 57) and intratumor leucocyte subsets (n = 14) at T3 versus T1 in 19 patients for whom both PBMC flow cytometry and tumor RNA-seq data were available. D, Representative correlations between individual patient values of the indicated PBMC and intratumor leukocyte subsets (P < 0.05, Benjamini–Hochberg FDR < 0.1) at T1 and T3.