Acetoacetate alters the colonic microbiota, expands intra-tumoral MAIT cells and inhibits colorectal cancer

The ketones beta-hydroxybutyrate (BHB) and acetoacetate (AcAc) have wide ranging effects on host and microbial physiology. Ketogenic diets and exogenous BHB can be pro- or anti-tumorigenic, but the potential for acetoacetate to alter anti-cancer immunity is unclear. AcAc can function as a signaling molecule and an energy source for host, microbial and tumor cells. We hypothesized that in vivo administration of AcAc will have both systemic and site-specific effects, impacting the stool microbiota, immune function, and tumor development.
Oral administration of an esterized form of AcAc (EAA) increased serum, fecal and tumor AcAc concentrations, and reduced tumor burden in two genetic models of colorectal cancer. In host cells, scRNA-Seq and flow cytometry identified changes in immune, tumor, epithelial, and stromal cells within the tumor microenvironment (TME). Mucosal associated invariant T (MAIT) cells were increased in treated tumors, and MAIT cell expression of IFNg, granzymes and perforins was higher than vehicle-treated controls. AcAc treatment also altered the microbiota. 16S rRNA gene amplicon analysis revealed enrichment of fecal Bifidobacteria following treatment. In vitro cultures supplemented with AcAc increased Bifidobacteria growth in a dose-dependent manner, while other taxa were not affected by supplementation. This suggests AcAc alters microbial composition by providing a growth advantage to specific taxa. Subsequently, metabolomics studies identified altered abundance of Bifidobacteria metabolites in multiple sites. In vivo stable isotope tracing of orally administered 13C EAA revealed 13C incorporation into a range of tumor metabolites including TCA cycle intermediates and amino acids.
Together, these results support that exogenous AcAc reduces neoplastic progression, alters colonic microbial composition, and promotes MAIT cell cytotoxicity. This reveals a connection between ketones, the microbiota and unconventional T cells. Metabolomics data, including results from in vivo 13C EAA stable isotope tracing, provided further insights about AcAc metabolism, and have informed mechanistic studies. The aims of ongoing work include determining how ketones regulate microbial metabolism and enhance anti-tumor MAIT functions. These studies will reveal metabolic processes that could inform ketone treatment or identify downstream immunometabolic pathways as novel therapeutic targets.