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

Abstract

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.

UFObow: A single-wavelength excitable Brainbow for simultaneous multicolor ex-vivo and in-vivo imaging of mammalian cells

“…29c ), suggesting that all cells in a clone originating from a single tumor cell are identical in color. Then, we monitored the growth dynamics of tumor clones in a liver metastasis mouse model 48 , 49 created by intrasplenic injection of UFObow-B16 cells.…”

“…The process of tumor cell colonization and colony formation in vivo was monitored through a liver window 48 , 50 for 9 days under an inverted confocal microscope equipped with a 405-nm laser (days 3, 5, 7, and 9) (Fig. 5a ).…”

Advances in intravital imaging of liver immunity using optical microscopy and labeling methods

“…This upregulation of ROS induced the activation of intracellular caspase‐3 (Figure 7), resulting in cell membrane perforation and the release of intracellular antigen tfRFP, which combined with an extracellular antibody to form an antigen–antibody complex. Further activation of the complement system accelerated and amplified the cell membrane perforation effect, while recruiting neutrophils and F4/80 + macrophages to the liver for synergistic antitumor effects [71].…”

“…In the process of tumor development, genetically programmed changes occur in tumor cells and surrounding cells [70] (tfRFP)-labeled B16 cells expressing apoptosis-related probes, including reactive oxygen species (ROS), caspase-3, and calcium probes. Employing this model, they observed the immune response in liver metastasis using intravital optical molecular imaging [71]. They found that, at the early stage, B16 tumor cells injected into hepatic sinuses via the spleen were influenced by blood flow shear forces and metabolic changes and that the ROS level was gradually increased.…”

“… Reactive oxygen species (ROS) elevation and caspase‐3 activation in tumor cells circulating in the liver [71]. (a) Representative intravital images of the liver in nonimmunized mice: top row, fluorescence images; middle row, HyPer7 ratio (F488/F405) images of MLS‐HyPer7‐B16 cells (stably expressing the mitochondria localized‐ROS probe); bottom row, C3 ratio (FRET/Cyan Fluorescent Protein) images of tfRFP‐C3‐B16 cells (stably expressing the caspase‐3 probe).…”