Adjuvant choice shapes macrophage activation and cytokine responses to freeze–thaw breast cancer antigens

To investigate how different adjuvants influence macrophage activation induced by freeze–thaw whole-cell lysates prepared from two breast cancer cell lines (MCF-7 and MDA-MB-231), we evaluated the effects of alum and saponin in J774 macrophages by assessing cell viability, nitric oxide (NO) production, and cytokine responses. Freeze–thaw breast cancer antigens (FTBA) triggered macrophage responses in a dose-dependent manner, with 40 µg/mL providing a suitable balance between activation and viability. When combined with adjuvants, FTBA + alum maintained higher macrophage viability compared with FTBA + saponin. Alum-adjuvanted FTBA induced stronger NO production than saponin-adjuvanted formulations. Cytokine profiling showed that alum-adjuvanted FTBA induced the strongest cytokine responses overall, with fold-increases in IL-6, IL-12, TNF-α, and GM-CSF observed for both antigen sources. Overall, alum enhanced macrophage activation with lower cytotoxicity than saponin, indicating that adjuvant selection significantly modulates innate immune responses to freeze–thaw tumor antigens.