In Vivo Optical Imaging The Immune Cells In The Tumor Microenvironment During Immunotherapy

The whole process of the “tumor immunotherapy” is like a black box, the scientists know much more information about the input(immunotherapy) and the output(tumor elimination). However, the relativeinformation for the cellular and molecular events occurred at what time and place during entire treatment process is still little known. Optical imaging with features of visualization, high spatial and temporal resolution, high sensitivity, dynamics and mutli-colordetections is widely used in the studies of tumor immunotherapy. The intravital optical imaging technology allows us to study the recruitment, migration and interaction of the immune cells in the tumor microenvironment to deeply study the related immunological mechanisms during the immunotherapy. In this study we used cyclophosphamide-adoptive cell(CTX-ACT) immunotherapy toexplore the “fight” between immune cells and tumor cells in the tumor microenvironmentin vivo. Although CTX-ACT combined immunotherapy had anti-tumor effect, how this treatment impact on the spatial and temporal changes of various immune cells, and how these changes further affect the immune effect, are still unclear, especially lack of behavior characteristics of various immune cells in the tumor microenvironment and use the information to characterize the immunological function and other aspects. Therefore, this study constructed amulti-color labeled tumor microenvironmentmodelandusedlarge-field intravital imaging technology to dynamically observe the key cellular events of the immune response induced by CTX-ACT immunotherapy. Furthermore, the improved immunotherapy schedulewhich was according to the results of intravital imaging obtained a desirable curative effect. This study achieved the following results:(1) Establisheda tumor-bearing mouse model with multi-color labeled tumor microenvironmentwhich is suitable for in vivo optical imaging. Based on multi-color fluorescent labeling technology, the large-field optical imaging and mouse model withwindow chamber, we observed the CFP labeled B16 tumor cells, Di R-BOA(near-infrared dye)labeled blood vessels, as well as GFP, RFP and YFP labeled various immune cells in the tumor microenvironment by intravital imaging. Our study provided an ideal mouse model to study the spatial and temporal dynamic changes of immune cells in the tumor microenvironment during the immunotherapy.(2) Real-time confocal imaging of the whole process of tumor-specific CTL(Cytotoxic T lymphocyte) killing tumor cells in vitro. Flow cytometry results showed that in vitro cultured tumor-specific CTL having strong anti-B16 tumor cell cytotoxicity. Real-time confocal imaging showed that after dozens of CFSE(green fluorescent dye) labeled CTL surrounding a group of CFP labeled B16 cells to contact with each other for 30 ~ 40 min, there was no significant changes inthe cell form of B16 cells, but the fluorescent signal of the B16 cells rapidly disappeared one by one, and the cells rapidly collapsed. This phenomenonwasvery similar to the "explosion" caused fast chain death. The imaging results suggest that, the response of CTL killing tumor cells in vitro is fast, efficient, andvisualized.(3) Studiedthe immune cell dynamic changes and mechanisms of curative effect induced bytumor immunotherapy by using in vivo optical imaging techniques. We observed the dynamic changes of m RFP labeled Tregs, CFSE labeled adoptive CTL and YFP labled DC in the tumor microenvironment after CTX-ACT combined treatment by long-term intravital dynamic imaging. The intravital imaging results showed that, Tregs accumulated at the tumor area at the early stage of tumor development, and when the solid tumor fromed, Tregs were around the the solid tumor and formed an "immunosppressive ring", which blocked the adoptive CTL migrating to the tumor area. The CTX treatment applied on 4 days after B16 tumor cells implantation deleted most of the Tregs of the tumor bearing mouse and suppressed Tregsforming the “immunosuppressive ring” around the solid tumor. In this way, the CTX treatment promoted the adoptive CTLs and mature DC recruiting to the tumor area and infiltrating into theparenchyma, which further induced effective anti-tumor immune response.(4) Studiedthe motility behaviors of various immune cells during the tumor immunotherapy. The intravital imaging results showed that after CTX-ACT combined therapy, the motility of the adoptive CTLs in details could be quantified over four stages. At the third stage(5 days after CTX-ACT treatment), the motility of CTL at both tumor periphery and deep parenchyma were rapidly increased, reaching 5.79 ± 3.12 ?m/min and 3.22 ± 2.58 ?m/min. Meanwhile, the anti-tumor effectreached the peak, and resultedin large-scale tumor death that consisted of tumor “shrinking” from the outside and “melting” from the inside. Twenty four hours after CTX-ACT treatment, theendogenous TII(Tumor infiltrating immunocytes) were transientlt activated. The average speed of TIIs reached to 4.92 ± 2.8 ?m/min, which was approximately 2.4-fold faster than the speed on the day before ACT treatment and 3.6-fold faster than48 h after CTX-ACT treatment. Meanwhile, most of the TII moved quickly towards the tumor parenchyma to further activate the subsequentanti-tumor immune response. Through intravital imaging of movement behavior of several immune cells(DC, CTL and TII), we found that the movement behavior of immune cells had two characteristics, which were "intermittent walk" and "Zigzag turning preference". Based on this, we established “Zigzag generalized Lévy walk” model to accurately depict the movement behavior of immune cells in vivo.In summary, this study established a tumor bearing mice model with multi-color labeled tumor microenvironment, and successfully obtained the dynamic information(such as the aggregation, spatial distribution, migration and cell contaction) of several immune cellsin the tumor microenvironment duringimmunotherapy through intravital imaging. The imaging results revealed the possible mechanism of CTX-ACT treatment to inhibittumor growth. The combined treatment blocked the formation of “immunosuppressive ring” around the solid tumor by Tregs, elicited a transient activation of tumor-infiltrating immunocytes with quick movement towards the tumor parenchyma, and accelerated the infiltration of adoptive T lymphocytes and dendritic cells. Therefore, the combination treatment of CTX and ACT has both endogenous and exogenous "synergistic enhancement" effect, which obtained the good curative effect.