Intravital molecular imaging reveals that ROS-caspase-3-GSDME-induced cell punching enhances humoral immunotherapy targeting intracellular tumor antigens

Tumor antigens (TAs)-induced humoral immune responses or TAs-specific antibodies have great application prospects for tumor therapy. However, more than half of TAs are intracellular antigens (intra-Ags) that are hardly recognized by antibodies. It is worthy to develop immunotherapeutic strategies for targeting intra-Ags. Methods: We used the far-red fluorescent protein tfRFP as an intracellular antigen to immunize mice and generated a liver metastasis model by injecting tfRFP-expressing B16 melanoma cells (tfRFP-B16) via the spleen. Intravital molecular imaging and atomic force microscopy were performed to visualize the formation of tfRFP antigen-antibody complexes (also known as immune complexes) and punched holes in cell membranes. Results: The results showed that the tfRFP-elicited immune responses inhibited the metastasis of tfRFP-expressing melanoma cells in the liver. In the circulating tfRFP-B16 tumor cells, elevated reactive oxygen species (ROS) induced slight caspase-3 activation, a probable key factor in the cleavage of gasdermin E (GSDME) proteins and punching of holes in the tumor cell membrane. Increased tumor cell membrane permeability led to the release of intra-Ag tfRFP and binding with anti-tfRFP antibodies. The formation of tfRFP antigen-antibody complexes on the membranes of tfRFP-B16 cells activated complement components to form membrane attack complexes to further destroy the cell membrane. Neutrophils were rapidly recruited, and F4/80⁺ macrophages phagocytized the dying tumor cells. Conclusion: The process of circulating tumor cell elimination in the tfRFP-immunized mice was triggered through the ROS-caspase-3-GSDME pathway to form intra-Ag-antibody immune complexes, which were involved in the activation of the complement system, as well as the recruitment of neutrophils and F4/80⁺ macrophages. An intra-Ag-elicited humoral immune response is a potent strategy for eliminating liver metastasis, which is unaffected by the liver immune tolerogenic status.