Fasting-mimicking diet remodels gut microbiota and suppresses colorectal cancer progression

Colorectal cancer (CRC) is a leading cause of cancer death globally, with diet playing a significant role in its progression. While calorie restriction and fasting show anti-tumor effects, adherence challenges limit their clinical use. Fasting-mimicking diets (FMDs) offer a more feasible alternative, inducing similar physiological responses while providing essential nutrients. Previous research demonstrates FMD's ability to suppress CRC progression by inducing cell apoptosis and suppressing aerobic glycolysis, and by boosting antitumor immune responses. However, the interplay between FMD, the gut microbiota, and the immune system in CRC remains poorly understood. This study aimed to investigate the impact of cyclic FMD on immune cell profiles and gut microbiota composition in a CRC mouse model, and to assess the synergistic effects of combining FMD with immunotherapy.

Existing literature highlights the link between dietary patterns and CRC development and progression. Calorie restriction and fasting have demonstrated anti-cancer properties in various studies, impacting tumor cell metabolism and immune responses. FMDs, designed to mimic the benefits of fasting while maintaining nutrient intake, have shown promise in preclinical models. Studies have shown FMD's ability to suppress CRC progression through mechanisms such as increased cell apoptosis and reduced aerobic glycolysis. Furthermore, FMD's impact on the immune system, specifically its potential to enhance antitumor immunity by modulating B cell class switching, is also under investigation. The combination of FMD with other therapies, like chemotherapy and immunotherapy, has demonstrated synergistic anti-cancer effects in various tumor models. However, a comprehensive understanding of FMD's impact on the gut microbiota and its interplay with the immune response in CRC is still lacking. The existing literature on FMD's effects on gut microbiota is limited, primarily focusing on inflammatory bowel disease (IBD).

This study utilized male C57BL/6 mice (6–8 weeks old) and employed three main mouse models: 1) FMD + CRC model: Mice received either a standard AIN-93G diet or a cyclic FMD (4 days FMD followed by 3 days of normal diet). Subcutaneous MC38 CRC cells were injected concurrently with the start of FMD. 2) CRC + bacteria tumor model: Mice received AIN-93G diet and were pre-treated with antibiotics to ensure microbiota consistency. They then received daily oral gavages of *Escherichia coli* (control), *Lactobacillus murinus*, *Lactobacillus johnsonii*, or a combination of both *Lactobacillus* species. 3) FMD + anti-PD-1 + CRC tumor model: Mice received either standard diet or FMD and were treated with IgG (control) or anti-PD-1 antibody every three days. Tumor growth was monitored, and various analyses were performed. Following euthanasia, tumor tissues and peripheral blood mononuclear cells (PBMCs) were collected for analysis. Flow cytometry was used to analyze immune cell populations (CD45⁺, CD8⁺ T cells, CD4⁺ T cells, NK cells, dendritic cells, and macrophages) in both tumor tissues and PBMs. Immunohistochemistry (IHC) was performed to assess Ki67 (cell proliferation) and CD31 (angiogenesis) expression in tumor tissues. Immunofluorescence staining was done to visualize CD8 in tumor tissues. Fecal samples were collected at the end of the final FMD cycle for 16S rRNA sequencing to assess gut microbiota composition. Quantitative PCR (qPCR) validated the abundance of specific *Lactobacillus* species. PICRUSt2 analysis predicted microbial functions from 16S rRNA data. Statistical analysis included unpaired t-tests, Mann-Whitney U-tests, one-way ANOVA, Welch ANOVA tests, Kruskal-Wallis tests, and linear correlation analysis, as appropriate. Ethical approval was obtained from the Clinical Research Ethics Committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine.

FMD significantly suppressed colorectal tumor growth, reducing tumor weight by 57% compared to the control group. FMD also decreased tumor cell proliferation (Ki67) and angiogenesis (CD31), without significantly affecting cell apoptosis. FMD increased the proportion of CD45⁺ cells in tumor tissue and CD8⁺ T cells in both tumor tissue and PBMCs. 16S rRNA sequencing revealed significant alterations in gut microbiota composition after FMD, with a five-fold increase in *Lactobacillaceae* and *Lactobacillus*. *Lactobacillus johnsonii* and *Lactobacillus murinus* showed the most significant increases. Oral administration of *L. johnsonii* mimicked FMD's anti-tumor effects, reducing tumor growth and increasing CD45⁺ and CD8⁺ T cells. Combining FMD with anti-PD-1 therapy showed a synergistic effect, further inhibiting tumor growth. Vancomycin treatment to deplete *Lactobacillus*, especially *L. johnsonii*, attenuated FMD's anti-tumor effects. PICRUSt2 analysis showed alterations in several KEGG pathways, including upregulation of naphthalene degradation and retinol metabolism and downregulation of tryptophan metabolism and the Foxo signaling pathway.

This study demonstrates that cyclic FMD effectively suppresses CRC progression in a mouse model. The observed reduction in tumor growth and angiogenesis, coupled with increased CD8⁺ T cell infiltration, indicates that FMD modulates the tumor microenvironment to promote anti-tumor immunity. The significant increase in beneficial gut microbiota, particularly *Lactobacillus johnsonii*, highlights the role of the gut microbiome in mediating FMD's anti-cancer effects. The findings that *L. johnsonii* supplementation alone mimics many of FMD's effects and that depleting *Lactobacillus* attenuates FMD's efficacy strongly suggest a crucial role for this bacterium in the anti-cancer mechanism. The synergistic effect of combining FMD with anti-PD-1 therapy suggests a potential strategy to improve immunotherapy effectiveness in CRC. This study provides strong evidence supporting the potential of FMD as a dietary intervention for CRC, particularly when combined with immunotherapies.

This study demonstrates that FMD suppresses CRC progression through modulation of both the immune system and the gut microbiota. *Lactobacillus johnsonii* appears to be a key mediator of FMD's anti-cancer effects. Combining FMD with anti-PD-1 therapy demonstrates a synergistic effect, enhancing tumor suppression. Future research should investigate the molecular mechanisms underlying FMD's interaction with *Lactobacillus* and explore the clinical potential of this dietary intervention in human CRC patients. Well-designed clinical trials are needed to validate these findings and determine the optimal application of FMD in combination with standard CRC treatments.

The study has some limitations. First, vancomycin treatment, used to deplete *Lactobacillus*, may also affect other gram-positive bacteria. Future studies could use specific bacteriophages for more targeted depletion. Second, while KEGG pathway analysis suggests a potential association with glycolysis, further investigation is needed to elucidate the downstream molecular mechanisms. Finally, the generalizability of these findings to human populations requires confirmation through larger-scale clinical trials. The potential for adverse effects due to the strict dietary regimen of FMD, such as malnutrition, should also be carefully considered in clinical applications.