Irradiated Liver Cancer Cell Vaccine Transfected With GM ‐ CSF Induces Specific and Long‐Lasting Anti‐tumour Immunity Through the Synergistic Effect of Oxidised mtDNA and GM ‐ CSF

Liver cancer remains a leading cause of cancer-related mortality worldwide, with limited durable responses to conventional therapies. Cancer vaccines utilising the immune system offer a promising alternative. Here, we developed a prophylactic whole-cell tumour vaccine by engineering Hepa 1-6 cells to express murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), and investigated its anti-tumour efficacy and underlying mechanisms. The Hepa 1-6-mGM-CSF vaccine provided complete and durable protection against both primary and rechallenge tumour inoculations. Hepa 1-6-mGM-CSF immunisation robustly activated dendritic cells (DCs) and T cells in both lymph nodes and spleen, characterised by enhanced DC maturation and migration, as well as the differentiation of T cells toward cytotoxic and memory phenotypes. Meanwhile, the Hepa 1-6-mGM-CSF vaccine remodelled the tumour microenvironment (TME) toward an immunostimulatory state. Critically, irradiation-induced oxidative stress in mitochondria promoted the release of oxidised mitochondrial DNA (ox-mtDNA), which subsequently activated the cGAS-STING signalling pathway. Ox-mtDNA synergized with vaccine-secreted GM-CSF to promote DC activation, antigen presentation and migration. In summary, our study demonstrates that the Hepa 1-6-mGM-CSF vaccine elicits robust anti-tumour immunity through the coordinated release of ox-mtDNA and GM-CSF, with ox-mtDNA synergistically enhancing immune activation via the cGAS-STING signalling pathway. Collectively, these findings highlight the Hepa 1-6-mGM-CSF vaccine as a promising strategy for liver cancer management.