Novel tryptophan 2,3-dioxygenase-targeted ruthenium(<scp>ii</scp>)-indole complex activates immunotherapy <i>in vitro</i> and <i>in vivo</i>

Immunotherapy targeting immune checkpoints has emerged as a promising strategy in cancer treatment; however, the heterogeneous and dynamic tumor microenvironment (TME) imposes critical constraints on therapeutic outcomes. Tryptophan 2,3-dioxygenase (TDO), often dysregulated in malignant tissues, plays a pivotal role in shaping an immunosuppressive milieu by depleting tryptophan, thereby hindering anti-tumor immune response. To counteract this immune evasion mechanism, we designed a novel indole-coordinated ruthenium(ii) arene complex (In-Ru), aimed at bolstering tumor immunotherapy and thwarting immune evasion by targeting TDO expression. Our findings reveal that In-Ru exerts markedly potent anti-proliferative effects against HepG2 cells. It achieves this by specifically localizing to the cell nucleus, inducing DNA damage, and initiating a cascade of necroptosis as well as immunogenic cell death (ICD), thereby potentially enhancing the immune system's capacity to recognize and attack cancer cells. RNA sequencing and qRT-PCR analysis indicate that In-Ru modulates pathways linked to tryptophan metabolism and immune reprogramming, with specific degradation of TDO protein and reversal of tryptophan-mediated immunosuppression. Furthermore, TDO inhibition boosts ROS production and induces necroptosis <i>via</i> mitochondrial damage, triggering a strong immune response. The tumor vaccine experiment revealed that In-Ru significantly reduced TDO levels and triggered ICD effect in liver cancer animal models. By reversing the immunosuppressive microenvironment, In-Ru facilitated the maturation of dendritic cells (DCs) and promoted T-cell infiltration, thereby achieving robust anti-tumor efficacy and long-lasting immune protection. This study represents the first report of a metal-arene complex with dual functions of TDO inhibition and ICD induction. It not only enhances anti-tumor immunogenicity but also effectively mitigates the risk of immune overactivation, offering a precise regulatory paradigm for the development of metal-based complexes in tumor immunotherapy.