A Flagellin-S100A9 Fusion Vaccine Breaks Immune Tolerance and Suppresses Experimental Lung Metastasis in Mice.

Lung metastasis is a major cause of cancer-related mortality, and the pre-metastatic niche (PMN) provides a window for early intervention. Here, transcriptomic profiling of pre-metastatic lungs in a subcutaneous B16-F10 melanoma model identified S100a8 and S100a9 as strongly upregulated PMN-associated genes. Exploratory TCGA analyses further linked high S100A8 or S100A9 expression with poorer overall survival in selected cancer types, although most associations were attenuated after multivariable adjustment. To evaluate these molecules as vaccine targets, we engineered recombinant fusion proteins linking mouse S100A8, S100A9, or tandem S100A8/A9 to a hypervariable-region-deleted flagellin (FliCΔHP) that retains Toll-like receptor 5 agonist activity. All three fusion proteins broke immune tolerance and elicited high-titer antigen-specific IgG responses, whereas only FliCΔHP-A9 significantly reduced experimental pulmonary metastasis in both male and female mice. Among the analyzed animals, anti-S100A9 IgG titers were inversely associated with metastatic burden. Immune serum inhibited S100A9-driven B16-F10 migration in vitro, and passive transfer of FliCΔHP-A9 immune serum reduced lung metastatic burden in naïve recipients, supporting an important contribution of humoral immunity to protection. Vaccination was also associated with lower pulmonary S100a8/S100a9 expression, reduced granulocytic and monocytic myeloid-derived suppressor cell accumulation, increased CD8+ T-cell frequencies, and an IFN-γ/IL-12-enriched pulmonary immune environment. Collectively, these findings identify S100A9 as an effective vaccine target in this experimental setting and suggest that S100A9-directed humoral immunity is accompanied by remodeling of the pulmonary immune microenvironment. Validation in spontaneous metastasis models and translational systems will be required to define the broader therapeutic potential of FliCΔHP-A9.