The role of tumor-associated neutrophils (TANs) infiltrating colorectal cancers remains debated. Here, we unravel that TAN recruitment and function are modulated by intra-tumoral microbiota, and only defined bacteria can unleash neutrophil cytotoxic potential.

Human CRC cell lines were injected intra-cecum into NSG mice, and chemokine expression was assessed by qPCR. Predominant CRC-associated bacteria, including Fusobacterium nucleatum (Fn) and Bacteroides fragilis (Bf), were cultured anaerobically and incubated with CRC cells or neutrophils from healthy donors and patients. Chemokine expression and secretion were measured by qPCR and ELISA. Neutrophil migration was assessed using transwell assays, and activation markers were analyzed by flow cytometry. Cytotoxic activity was evaluated by CRC cell viability after exposure to neutrophil supernatants. TAN phenotypes were profiled in freshly resected CRC and matched normal tissues. To assess the clinical relevance of Fn–TAN interactions, we analyzed the prognostic value of neutrophil infiltration and Fn load in a well-characterized CRC tissue microarray and in the TCGA dataset.

Fn promotes production of neutrophil-recruiting chemokines by tumor cells and enhances neutrophil migration more efficiently than BF. Importantly, Fn, but not Bf, triggers neutrophils to release cytotoxic proteins showing tumoricidal activity in vitro and in xenograft models. Mechanistically, these effects are elicited upon Fn binding to an activating receptor (aR) expressed by neutrophils but are impaired upon aR blockade or loss-of-function polymorphism. Consistently, in human CRCs elevated Fn loads and high TANs densities correlate with improved prognosis, whereas lack of aR expression is associated with reduced patient survival.

Our findings identify microbiota composition and host genetic background as critical determinants of neutrophil functional profiles and offer insights into neutrophil-targeted therapeutic strategies in CRC.