Activation of an adaptive antitumor immune response by the polymeric fluoropyrimidine CF10 involves TS/Top1 dual targeting.

5-Fluorouracil (5-FU)-based regimens remain the backbone of therapy for metastatic colorectal cancer (mCRC), yet durable responses are rare.CF10, a next-generation polymeric fluoropyrimidine, has demonstrated superior antitumor activity compared with 5-FU in preclinical models. Here, we evaluated whether CF10 more effectively induces immunogenic cell death (ICD) and promotes antitumor immunity, while characterizing its dual mechanism involving thymidylate synthase (TS) inhibition and replication stress through Topoisomerase 1 cleavage complex (Top1cc) stabilization and γ-H2AX accumulation. In murine (MC38) and human (HCT116) colorectal cancer cells, CF10 induced significantly higher levels of ICD markers-extracellular ATP, HMGB1 release, and surface calreticulin-than 5-FU and triggered robust Top1cc stabilization with γ-H2AX foci formation. Conditioned media from CF10-treated cells enhanced dendritic cell (DC) maturation and secretion of TNF-α, IL-1β, CCL2, and CCL4. In vivo, CF10 treatment in C57BL/6 mice bearing orthotopic MC38 liver metastases increased CD4⁺, CD8⁺, and γδ T-cell infiltration, reduced myeloid-derived suppressor cells (MDSCs), and decreased hepatic tumor burden. CF10 also decreased FoxP3⁺ regulatory T cells and CD206⁺ immunosuppressive macrophages. DC vaccination using CF10-conditioned supernatants modestly extended survival and increased tumor-infiltrating T cells compared with 5-FU or untreated controls. CF10 elicits a dual-action mechanism-cytotoxicity via TS inhibition and Top1cc-dependent replication stress (γ-H2AX) and immunogenicity through ICD induction-driving potent DC activation and adaptive immune responses. These findings position CF10 as a promising immunomodulatory chemotherapeutic for mCRC and support clinical evaluation, including combination strategies with immune checkpoint blockade.