Intravital Imaging Of T Cell-Induced Tumor-Related Molecular Events And T Cell Killing Efficiency

Intravital optical molecular imaging techniques can not only track the migration,aggregation and contact of cells in vivo,but also visually presents the dynamic changes of intracellular molecular signals,providing powerful tools for studying cell functions in vivo.Tumor immunotherapy uses the characteristic of the immune system to recognize and eliminate tumors to treat tumors,and shows unique advantages in clinical practice.Cytotoxic T lymphocytes(CTLs)are the main effector cells that kill tumor cells in the immune system and play a key role in the process of tumor elimination.As an organ with unique immune characteristics,it is a high-risk organ of hepatocellular carcinoma and cancer metastasis.However,in the immune-tolerant microenvironment of the liver,the dynamic information of CTL-induced tumor cell-related molecular events and the efficiency of CTL to kill tumor cells are not clear.The answers to these questions will help understand the mechanism of CTLs clears tumor cells in vivo.Intravital imaging was used to monitor the dynamic process of CTL specific elimination of solid tumors.The capacity of CTLs to eliminate tumor cells in the liver and spleen was quantitatively compared,and the timing of CTL-induced calcium signaling and apoptosis signaling of tumor cells was revealed.The main findings are as follows:(1)Intravital imaging monitored the movement characteristics of the CTLs in the tumor microenvironment after adoptive cell therapy.The results showed that CTLs speed reduced,more tortuous trajectories,and longer resting time when tumor cells expressing homologous antigen,whether it is primary tumors or liver metastases.These movement behaviors helped CTL to recognize and kill tumor cells.(2)The visualization model for dynamic characterization of tumor cell proliferation and apoptosis in vivo was established.A series of B16 tumor cell lines stably expressing genetically encoded molecular probes were screened,which was used to optically characterize the dynamic changes of tumor cell mitosis(H2B-m Cherry),apoptosis(apoptosis probe C3),and calcium signal(calcium probe Twitch2B).Furthermore,a multi-color labeled model in vivo was established,which was used to study the proliferation rate of tumor cells in vivo,to monitor CTL specific recognition and killing of tumors,and to intravital molecular imaging related molecular events.(3)According to the intravital imaging results of the CTL killing-induced tumor cell apoptosis,the capacity of CTL to kill tumor cells in vivo was quantitatively calculated.The quantitative data showed that each CTL killing liver metastatic tumor cells is 1.24 ± 0.11 tumor cells per day,which was less efficient than that in the spleen(3.18 ± 0.26 tumor cells/CTL/day).The killing efficiency of CTLs is restrained in the liver,which was related to the high expression of the immunosuppressive factor IL-10 in the liver.The capacity of CTLs to kill tumor cells in the liver was significantly improved after IL-10 blockade.(4)Intravital molecular imaging of calcium flux and apoptotic signals in tumor cells determined the timing of CTL-induced molecular events in tumor cells.CTL killing tumor cells led to a prolonged(> 30 s)calcium influx in tumor cells.The time of calcium influx occurred dozens of minutes before caspase-3 activity in tumor cells,implying that CTLs elicited prolonged calcium influx in tumor cells as a prerequisite step for tumor cell apoptosis.In summary,this article uses intravital molecular imaging to study the temporal and spatial characteristics of movement behavior and key molecular events in the process of CTLs killing tumors in vivo.A method for quantitatively evaluating the capacity of CTL to kill tumor cells in vivo was developed.CTLs killing tumors was restrained in the liver.CTL induced prolonged calcium influx of tumor cells may be one of the important factors that trigger the activation of caspase-3.This study provided a new method for visually evaluating tumor immunotherapy in vivo,which helps to reveal the bottleneck problem of the application of CTL in the treatment of solid tumors.