Nanostructured branched Y-DNA promotes antitumor immunity through dual activation of cGAS/STING and TLR9

Effective activation of innate and adaptive immunity remains a major challenge in cancer immunotherapy. In this study, we aimed to develop an immunostimulatory oligonucleotides for antitumor immunotherapy. We designed branched Y-shaped DNA (Yb-DNA) nanoparticles complexed with a reducible polycationic carrier (RPC-bPEI_0.8kDa), which we named YbNano, to enhance intracellular delivery and immune activation. The immunostimulatory activity of YbNano was determined in mouse bone marrow-derived dendritic cells. The efficacy of YbNano for antitumor immunotherapy was investigated using a melanoma allograft mouse model and a lung metastasis of breast cancer mouse model. YbNano exhibited uniform particle morphology and efficient cellular uptake in dendritic cells. YbNano induced robust expression of type I interferon (IFN-β), interleukin-12 (IL-12), chemokines such as CXCL10, and costimulatory molecules (CD80, CD86) in dendritic cells, promoting CD8⁺ T cell differentiation. In preclinical cancer models, intravenous administration of YbNano potentiated the efficacy of anti-PD-L1 therapy in B16F10 melanoma-bearing C57BL/6 mice and enhanced the therapeutic outcomes of doxorubicin or anti-PD-L1 treatment in suppressing lung metastasis in 4T1 breast cancer-bearing BALB/c mice. YbNano functions as a dual activator of cGAS/STING and TLR9, orchestrating dendritic cell activation and amplifying downstream innate and adaptive immune responses in tumor microenvironment. Collectively, these findings suggest that YbNano represents a rationally engineered, multifunctional nucleic acid-based immunotherapeutic agent with the potential to modulate the tumor microenvironment and to augment responses when used in combination cancer therapy.