Intravital Optical Imaging Investigation The Effect And Mechanism Of Intracellular Antigen-induced Immune Clearance Of Liver Metastasis

The tumor antigen-induced immune response and antibodies targeting tumor antigens play an important role in the antitumor effect,and the development of tumor vaccines based on these tumor antigens is one of the important aspects of tumor immunotherapy.Tumor antigens are divided into intracellular antigens（intra-Ags）and extracellular antigens.Due to the barrier of the cell membrane,intra-Ags cannot be directly recognized and bound by antibodies.One of the keys to tumor immunotherapy targeting intra-Ags is to change the permeability of cell membranes,thereby improving the recognition and binding of antibodies to intra-Ags.Molecular signaling changes in tumor cells(such as ROS,Ca²⁺,and cleavage of GSDME by caspase-3)are closely related to the permeability of cell membranes.However,the mechanism by which tumor molecular signaling affects the permeability of cell membranes remains unclear.Therefore,intravital optical molecular imaging to observe the molecular signal changes of tumor cells and study the molecular mechanism of improving the binding of antibodies to intra-Ags in tumor cells will contribute to the development and application of tumor vaccines targeting intra-Ags.In this paper,the liver with the characteristics of inducing immune tolerance and a high incidence of tumor metastasis was selected as the research subject.Combined with tumor cell strains that stably express genetically encoded molecular probes,the immunization of intra-Ags induced immune clearance of liver metastases was observed by intravital optical molecular imaging,and the molecular mechanism that promotes antibody binding to intra-Ags during the formation of liver metastases was revealed.The main contents and results are as follows:（1）A triple-fixed liver window model was established,in which the mouse,liver and liver window ring were fixed to minimize liver jitter during long-term intravital imaging,avoiding out-of-focus imaging.By analyzing the parameters of T cells’motility,it was revealed that subcutaneous immunization with antigens could reverse the changes in the motility parameters of antigen-specific CD4⁺T cells and Treg cells caused by antigen presentation in the liver.（2）A tumor cell strain stably expressing ROS probes was constructed,and combined with the tumor cell strains expressing other molecular probes,the dynamic monitoring of ROS,caspase-3,and Ca2⁺signals of tumor cells in the process of liver metastasis was realized by intravital optical molecular imaging.The results showed that when the tumor cells arrived at the liver,the tumor cells underwent periodic calcium fluctuations and ROS elevation.Caspase-3 was activated 10 minutes after tumor cells arrived.（3）Using intracellularly expressed tetrameric far-red fluorescent protein（tf RFP）as an intracellular antigen model,intravital optical imaging was used to study the immune clearance of tumor cells induced by intra-Ags.In the process of immune clearance of liver metastases,intra-Ags released from tumor cells bind to antibodies in the tissue environment,forming antigen-antibody complexes on the cell membrane that can activate the complement to form C5b-9⁺membrane attack complexes that further damage the cell membrane and release more intra-Ags,while neutrophils are recruited around the metastases.（4）In the process of immune clearance of liver metastases induced by intra-Ags,intravital optical molecular imaging revealed that ROS increased in some tumor cells in the early stage as they entered the hepatic sinusoids,and the increase in ROS led to the activation of caspase-3 in some cells,which in turn induced the cleavage of GSDME.Elevated ROS and the cleaved N-terminal GSDME create holes in the cell membrane,resulting in the release of intra-Ags.Thus,these results suggest that tumor cells initiate intra-Ags-induced immune clearance of liver metastases by triggering the ROS-caspase-3-GSDME pathway.In conclusion,an intravital optical molecular imaging model of the liver was established,revealing the process of intra-Ags induced immune clearance of liver metastases and the changes of molecular signals in tumor cell.This study found that ROS-caspase-3-GSDME pathway-induced cell punching is a key step for antibodies to recognize intra-Ags.Antigen-antibody complexes,the complement system,neutrophils and hepatic macrophages play an important role in the clearance of liver metastases by intra-Ags-induced immune responses.This study provides new ideas and strategies for the development of tumor vaccines and antibody therapy against intra-Ags.