Caerin 1.1/1.9 peptides cooperate with 5-fluorouracil to suppress melanoma by reprogramming myeloid suppressor cells and regulatory T cells

Purpose Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) are major barriers to effective anti-tumour immunity in melanoma. While 5-fluorouracil (5-FU) can partially modulate tumour immunity, its effects are often incomplete. We investigated whether combining host-defence peptides caerin 1.1/1.9 (F1/F3) with 5-FU enhances antitumour efficacy by reprogramming immunosuppressive compartments within the tumour microenvironment (TME). Experimental Design: Antitumour activity of F1/F3 and 5-FU was assessed in the B16 melanoma model using survival, tumour burden, and immune profiling analyses. Cytotoxic interactions were evaluated in vitro . Flow cytometry, single-cell RNA sequencing, T-cell receptor repertoire analysis, cytokine assays, and bioinformatic approaches characterised immune composition, functional states, and intercellular communication under different treatments. Results F1/F3 and 5-FU did not exhibit synergistic cytotoxicity in vitro but produced marked therapeutic synergy in vivo . Dual therapy (DT) significantly prolonged survival and enhanced intratumoural CD8⁺ T-cell infiltration while reducing immunosuppressive MDSCs and Tregs. scRNA-seq revealed that DT selectively impaired metabolically active neutrophil-like PMN-MDSCs and reprogrammed monocytic M-MDSCs toward an IL-1/NF-κB–driven inflammatory, immune-interactive state. Tregs under DT showed destabilisation of canonical suppressive programs and acquisition of effector-like features. These changes were accompanied by enhanced myeloid–lymphoid communication and pronounced clonal focusing of tumour-infiltrating T cells, indicating antigen-driven immune activation. Conclusions F1/F3 peptides convert 5-FU from a partially immunomodulatory chemotherapy into a potent immune-reprogramming regimen by dismantling myeloid and Treg-mediated suppression. This coordinated remodelling of the TME promotes effective adaptive immunity and provides a mechanistic rationale for integrating host-defence peptides with chemotherapy to overcome immune resistance in melanoma.