Immunological Mechanism Of Sonodynamic Therapy For The Treatment Of Liver Cancer In Mice

Sonodynamic therapy(SDT)is a new type of cancer treatment,its basic principle of is similar with photodynamic therapy(PDT).Sensitizers can specifically concentrate on the tumor tissue,the radiation of acoustic with specific parameters can make the oxygen contained in organization change into reactive oxygen species(ROS),and produce oxidation reaction with tumor cells,which eventually lead to death of tumor cells.It is worth mentioning that ultrasound can go deeply into the organization comparing with photodynamic therapy(PDT),which have poor tissue penetration.Some literatures showed that the penetration depth of laser light to the organization is 1 to 2 cm,while the penetration depth of ultrasonic can reach more than 5 cm.It is possible that the use of SDT to make a treatment of deep tumors.At present,the preclinical studies of SDT have received some encouraging results,but there is no uniform standard in the ways of selections of different parameters and different levels,which make the best curative effect of SDT tumor treatment couldn't be really play out.The results of SDT experiments in vitro have not strongly persuasion,so this may be an important reason why SDT has not really been applied in clinical practice.Recent studies have reported that tumor treatment with PDT has the characteristics of tumor vaccine.We hypothesize that,similar to PDT:the same mechanism and excitation principle,SDT may perform effectively as cancer vaccines.Thus,we developed a therapeutic strategy to explore whether SDT can eliminate primary tumors,inhibit metastases,and prevent tumor relapse.Objective To study the abscopal effect of sonodynamic therapy(SDT)treating liver cancer in mice.Methods To investigate the efficacy of SDT,the four parameters:photosensitizer concentration,ultrasound intensity,irradiation time,and ultrasound frequency,were set at three levels.An orthogonal experimental approach was adopted using these four parameters.These three levels of four factors were divided into nine groups.MTT analysis,cell apoptosis,intracellular ROS in vitro,and anticancer efficacy in vivo were conducted to explore the optimal parameters for SDT.And the optimal parameters will be used in subsequent experiments.Next,we explored the immunological mechanism of sonodynamic therapy in vitro and vivo.The expression of calreticulin(CRT)on the surface of the tumor cells were measure by using immunofluorescence and flow cytometry.We established H22 tumor model in mice to observe SDT inhibition effect of H22 mice tumor size and used ELISA to study the levels of IL-2,IFN-y and IL-10 in serum of four groups.Using Immunohistochemical experiments were used to detect the expressions of CD4,CD8,CD68,CD163,and FoxP3 in tumor tissues.Then we did the LDH release experiments to evaluate treatment specificity of spleen cells in all groups to H22 tumor cells and S180 tumor cells.Immunohistochemical experiments were used to confirm the tumor immune specificity of SDT in distant tumor tissues.Finally,rechallenging mice whose distance tumor were completely disappeared(abscopal effect)with the same number live H22 cells to observe the tumor growth.Results According to the results of the MTT assay,the ideal parameters were A2,B2,C3,and D3;The percentage of apoptotic cells of flow cytometry results showed that the ideal parameters were A3,B3,Cl,and D3;The generation of ROS demonstrated that the effect of SDT was primarily determined by the concentration of HPD.For HPD concentration,A3 was the most effective.Therefore,the efficacy of SDT is optimal with 20?g/ml HPD.Conclusively,the optimal parameters of SDT:ultrasonic intensity of 1.0 W/cm2,sensitizers concentration of 20?g/ml,irradiation time for 30 minutes.Due to the ultrasonic frequency has no obvious difference among the three levels,so for ultrasonic frequency in the subsequent experiments we choose to use 50%.The curative effect of single ultrasonic radiation treatment is weak,so we choose continuous irradiation four cycles(that is,the irradiation time is 2 hours).The results of immune effect of SDT were shown as following:The flow cytometry and immunofluorescence showed that the SDT group was detected more significantly expressions of CRT than control group and HPD group in H22 cell surface.SDT group could be observed a obvious inhibition of tumor growth,the tumor became smaller along with the time extension.At the same time,the treatment effect of six cycles is more obvious than that of four cycles,so 6 cycles was used for the follow-up experiments.The level of IL-2,IFN-y were obviously higher than the corresponding cytokine levels of the other three groups(P<0.0001)in mice serum.The level of IL-10 was significantly lower than other groups(P<0.0001).In the tumor tissues of SDT,the expression of CD4,CD8,and CD68 were higher,while a low level of CD 163 and FoxP3 was showed.Single factor variance analysis showed that:significant difference between groups(CD4(P<0.001);CD8(P<0.001);CD68(P<0.001);CD163(P=0.005);And FoxP3(P<0.0001).SDT group had the effect of inhibiting the distant tumor growth.As shown in results,for H22 tumor cells,the ability of CTL cells of SDT group was obviously higher than that of the other three groups(P<0.0001).When E:T ratio was 100:1(P<0.0001),CTL cells had the strongest ability to kill tumor cells.On the contrary,for S180 tumor cells,the killing capability of CTLs cells had no significant difference between four groups(P=0.123).A strong infiltration of CD4,CD8 T cell could be found in SDT group in distant tumor tissues which were quantified based on IOD values by using Image-Pro Plus 6.0(CD4 P<0.0001;CD8 P<0.0001).Conclusion SDT have the potential of producing effective cancer vaccines and the inhibition of tumor growth in primary and distant,it deserves further research.