Experimental Study On The Antitumor Immunity Following Microwave Thermal Ablation In Tumor-bearing Mice

Objectives To explore the patterns of the heat shock proteins(HSPs) expression in tumor cells following microwave thermal ablation in tumor-bearing mice; the variations of the antigen presenting cells around the tumor, and the expression of the major histocompatibility complex(MHC) H-2K~b/H-2D~d and I-A~d molecules as the first signals of antigen specificity and CD86 molecule as the second signals of co-stimulatory molecules which are indispensable for T cell activation; meanwhile, the effects of tumor cell suspension treated with microwave thermal ablation on the antigen presenting functions of macrophages are also to be investigated.Materials and Methods (1)Experiments of histology: The mice of H22 transplantation tumor under the skin were divided into experimental and sham groups. After etherization, the microwave antenna was inserted the center of tumor along the long axis. The experimental group treated with 20W by microwave thermal ablation, and continued 120 second, and the temperature aside the center 5mm were measured in partly mice which reached 50°C above quickly. In the sham group, mice were treated exactly as in experimental group with the exception that the microwave was not activated. Eight mice of each group were randomly killed after 6h, 12h, 18h, and 24h after the experiment, and the tumor nodules were resected quickly under the sterility condition. The patterns of the HSP27, HSP60, HSP70 and HSP90α expression in tumor cells following microwave thermal ablation were observed by the PowerVision two-step immunohistochemical results, and the time-dependent alteration of cellular distribution of HSPs were also analyzed. The numbers varied of antigen presenting cells which the expression of MD-1 molecule were positive. The expression of the MHC H-2K~b/H-2D~d and I-A~d molecules and the CD86 moleculewere analyzed in the peripheral area around tumor nodules. The status of CD4 and CD8 lymphocytes infiltration were analyzed too. (2) Experiments of ctyology: The macrophages were harvested from abdominal cavity of mice. Experimental groups were co-incubated together with H22 tumor cells suspension 0.5 ml (approximately 5×10~6 cells) treated with 50W600s microwave thermal ablation which were directly added into the culture system. The control groups were co-incubated macrophages with the same numbers of untreated H22 tumor cells, and the placebo groups were replaced tumor cells with saline solution. The experiments were ended after 9h, 18h, 27h, 36h, and 45h. The expressive intensities of the molecules of MD-1, MHC H-2K~b/H-2D~d , MHC I-A~d and CD86 on macrophages were analyzed, and the morphologic changes of macrophages were also observed. Moreover, the survival time of mice of rechallenged by the same tumor cells between the mice immunized by H22 tumor cells treated with microwave thermal ablation and the mice of placebo control were investigated.Results (1) The results of the expression of HSPs: Before microwave thermal ablation, the H22 tumor cells also had a low level expression of HSPs mainly in cytoplasm, and the expressive intensities of HSP60 were the highest. After microwave thermal ablation, most of tumor cells around the area of necrosis center had significantly increased in the expressive intensities of HSP27, HSP60, HSP70 and HSP90α. In contrast to sham group, the expressive intensities of all the HSPs were higher (P<0.001 ), and the persistent time were longer. The numbers of membrane positive of HSP27,HSP70 and HSP90α were increased excepting HSP60. Analysis the peaking of expressive intensities, the peaking time of HSP27 and HSP60 were 24h, HSP70 were 12h, and HSP90a were 18h.( 2) The results of the antigen presenting cells infiltrating and the expression of the molecules related with immunity: The numbers of positive cells for MD-1, MHC H-2K~b/H-2D~d and I-A~d, CD86 molecules expression were increased among the peripheral area of tumor following microwave thermal ablation. Moreover, it was increased around peripheral tumor tissue, especially, the bigger professionalantigen presenting cells (positive for MD-1). (3) The results of the function of macrophages about the antigen presenting: The expressive intensities of experimental groups of macrophages in MD-1, MHC H-2K~b/H-2D~d and I-A~d, CD86 molecules were all significant increased, and among them, the intensities of MHC H-2K~b/H-2D~d and I-A~d molecules were highest in 9h, and MD-1 and CD86 molecules in 18h. However, the cells survival of experimental groups of macrophages were decreased significant with the activation, so that the numbers of macrophages were significant depleted in 36h and 45h. The macrophages of control groups nearly all died from 9h. The survival time of mice immunized by H22 tumor cells treated with microwave thermal ablation when rechallenged the same tumor cells were significant higher than the survival time of placebo control mice(P=0.026).Conclusions Microwave thermal ablation treatment can significantly prompt the expression of HSPs in tumor cells, and has a tendency of expression more in membrane. It also can significantly prompt the infiltrating of the antigen presenting cells in peripheral tissue of tumor, and increase the expression of the first signals of antigen specificity MHC molecules and the second signals of co-stimulatory molecules CD86 which are indispensable for T cell activation. The tumor cells suspension treated with microwave ablation can activate the function of macrophages about antigen presenting, and significantly prompt the expression of MHC molecules and CD86 molecules of it.