Indomethacin exerts both cyclooxygenase inhibition-dependent and independent mechanisms to enhance chemo-immunotherapy in mice.

Nonsteroidal anti-inflammatory drugs (NSAIDs) primarily act by inhibiting cyclooxygenases (COX1 and COX2), thereby reducing production of the proinflammatory mediator prostaglandin E₂ (PGE₂). Because PGE₂ is a critical driver of cancer progression and tumor immune evasion, this has motivated interest in combining NSAIDs with chemotherapy or immunotherapy for cancer treatment. However, since COX and PGE₂ levels vary across tumor types, it remains unclear whether tumor PGE₂ abundance solely dictates tumor response to NSAID-based therapies. Here, we investigated the therapeutic potential of indomethacin (Indo), a prototypical NSAID, in combination with cyclophosphamide (CTX), a widely used chemotherapeutic agent with immunostimulatory properties. Metronomic administration of Indo significantly enhanced the antitumor efficacy of CTX in multiple murine tumor models exhibiting variable COX2 and PGE₂ levels, including CT26, MC38, 4T1 and A20. The antitumor effects of CTX+Indo required CD8⁺ T cells and T-cell trafficking from tumor-draining lymph nodes and were further potentiated by anti-PD-1 blockade. Single-cell RNA sequencing (scRNA-seq) revealed that responsive CT26 tumors exhibited a reprogrammed tumor immune microenvironment (TIME), marked by increased effector CD8⁺ T-cell infiltration, reduced immunosuppressive myeloid populations, and enhanced interferon signaling in tumor cells. Importantly, Indo retained therapeutic benefit following CTX even in tumors incapable of producing PGE₂, demonstrating a critical contribution of COX-independent mechanisms, particularly inhibition of tumor-intrinsic oncogenic RAS signaling, to the enhanced efficacy of the CTX+Indo combination. Collectively, our results provide strong preclinical rationale for leveraging the COX/PGE 2 and RAS dual inhibitory capacities of NSAIDs to enhance the efficacy of chemotherapy and immunotherapy.