Abstract

Introduction Gut microbiota-derived metabolites play pivotal roles in clinical tumor treatment and progression. Cyclic di-AMP serves as both a bacterial signaling molecule and an immune activator of the STING pathway. However, knowledge on cyclic di-AMP production by gut commensals remains limited, hindering the rational application of gut bacteria in cancer therapy. Objectives This study systematically characterizes the metabolic profiles and genotypes associated with cyclic di-AMP synthesis in human gut commensals. We further validate the immune-activating and anti-tumor effects of high cyclic di-AMP-producing probiotics in both in vitro and in vivo non-small cell lung cancer models. Methods Cyclic di-AMP levels (intracellular and extracellular) were quantified via LC-MS in 51 representative gut bacterial species derived from 442 strains isolated from 119 human fecal samples. STING pathway activation was assessed by co-culturing THP-1 cells with supernatants from high cyclic di-AMP-producing gut probiotics. Anti-tumor efficacy was evaluated in a non-small cell lung cancer mouse model. Results Screening of 51 gut bacterial species identified 24 high cyclic di-AMP producers, with 18 exhibiting robust secretion capacity. Bioinformatic annotation revealed genes governing cyclic di-AMP synthesis, degradation, and secretion. Two food-grade probiotics, Limosilactobacillus fermentum DA785 and Lacticaseibacillus rhamnosus R7970, demonstrated efficient cyclic di-AMP secretion. Their supernatants significantly upregulated STING pathway-related gene expression and IFN-β secretion in THP-1 cells. Oral administration of these strains suppressed tumor growth in mice by activating immune responses within the tumor microenvironment. And Limosilactobacillus fermentum DA785 suppresses tumor growth via the STING pathway. Conclusion This study highlights the therapeutic potential of food-grade probiotics with high cyclic di-AMP production to augment anti-tumor immunity, off